Compositions for modulating C9ORF72 expression

ABSTRACT

Disclosed herein are compositions and methods for reducing expression of C9ORF72 mRNA and protein in an animal. Such methods are useful to treat, prevent, ameliorate, or slow progression of neurodegenerative diseases in an individual in need thereof.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0269USSEQ_ST25.txt created Apr. 15, 2016, which is 104 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD

Provided are compositions and methods for modulating expression ofC9ORF72 mRNA and protein in cells and animals. Such compositions andmethods are useful to treat, prevent, ameliorate, or slow progression ofneurodegenerative diseases, including amyotrophic lateral sclerosis(ALS), frontotemporal dementia (FTD), corticobasal degeneration syndrome(CBD), atypical Parkinsonian syndrome, and olivopontocerebellardegeneration (OPCD).

BACKGROUND

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative diseasecharacterized clinically by progressive paralysis leading to death fromrespiratory failure, typically within two to three years of symptomonset (Rowland and Shneider, N. Engl. J. Med., 2001, 344, 1688-1700).ALS is the third most common neurodegenerative disease in the Westernworld (Hirtz et al., Neurology, 2007, 68, 326-337), and there arecurrently no effective therapies. Approximately 10% of cases arefamilial in nature, whereas the bulk of patients diagnosed with thedisease are classified as sporadic as they appear to occur randomlythroughout the population (Chio et al., Neurology, 2008, 70, 533-537).There is growing recognition, based on clinical, genetic, andepidemiological data, that ALS and frontotemporal dementia (FTD)represent an overlapping continuum of disease, characterizedpathologically by the presence of TDP-43 positive inclusions throughoutthe central nervous system (Lillo and Hodges, J. Clin. Neurosci., 2009,16, 1131-1135; Neumann et al., Science, 2006, 314, 130-133).

To date, a number of genes have been discovered as causative forclassical familial ALS, for example, SOD1, TARDBP, FUS, OPTN, and VCP(Johnson et al., Neuron, 2010, 68, 857-864; Kwiatkowski et al., Science,2009, 323, 1205-1208; Maruyama et al., Nature, 2010, 465, 223-226; Rosenet al., Nature, 1993, 362, 59-62; Sreedharan et al., Science, 2008, 319,1668-1672; Vance et al., Brain, 2009, 129, 868-876). Recently, linkageanalysis of kindreds involving multiple cases of ALS, FTD, and ALS-FTDhad suggested that there was an important locus for the disease on theshort arm of chromosome 9 (Boxer et al., J. Neurol. Neurosurg.Psychiatry, 2011, 82, 196-203; Morita et al., Neurology, 2006, 66,839-844; Pearson et al. J. Nerol., 2011, 258, 647-655; Vance et al.,Brain, 2006, 129, 868-876). The mutation in the C9ORF72 gene is the mostcommon genetic cause of ALS and FTD. The ALS-FTD causing mutation is alarge hexanucleotide (GGGGCC) repeat expansion in the first intron ofthe C9ORF72 gene (Renton et al., Neuron, 2011, 72, 257-268;DeJesus-Hernandez et al., Neuron, 2011, 72, 245-256). A founderhaplotype, covering the C9ORF72 gene, is present in the majority ofcases linked to this region (Renton et al., Neuron, 2011, 72, 257-268).This locus on chromosome 9p21 accounts for nearly half of familial ALSand nearly one-quarter of all ALS cases in a cohort of 405 Finnishpatients (Laaksovirta et al, Lancet Neurol., 2010, 9, 978-985).

A founder haplotype, covering the C9ORF72 gene, is present in themajority of cases linked to this region.

There are currently no effective therapies to treat suchneurodegenerative diseases. Therefore, it is an object to providecompositions and methods for the treatment of such neurodegenerativediseases.

SUMMARY

Certain embodiments provide methods, compounds, and compositions forinhibiting expression of C9ORF72 mRNA and protein in cells, tissues, andanimals. Certain embodiments provide methods, compounds, andcompositions for reducing C9ORF72 mRNA and protein levels in cells,tissues, and animals. Certain embodiments provide antisense compoundstargeted to a C9ORF72 nucleic acid. In certain embodiments, theantisense compounds are modified oligonucleotides. In certainembodiments, the modified oligonucleotides are single-stranded.

In certain embodiments, C9ORF72 associated Repeat Associated Non-ATGTranslation (RAN translation) products are reduced. In certainembodiments, the C9ORF72 associated RAN translation products arepoly-(glycine-proline), poly-(glycine-alanine), andpoly-(glycine-arginine). In certain embodiments, certain C9ORF72 mRNAvariants are preferentially reduced. In certain embodiments, the C9ORF72mRNA variants preferentially reduced are variants processed from apre-mRNA containing intron 1. In certain embodiments, intron 1 containsa hexanucleotide repeat expansion. In certain embodiments, the C9ORF72mRNA variant preferentially reduced is a C9ORF72 pathogenic associatedmRNA variant. In certain embodiments, the C9ORF72 pathogenic associatedmRNA variant is NM_001256054.1 (SEQ ID NO: 1). In certain embodiments,the hexanucleotide repeat expansion is associated with a C9ORF72associated disease. In certain embodiments, the hexanucleotide repeatexpansion is associated with a C9ORF72 hexanucleotide repeat expansionassociated disease. In certain embodiments, the hexanucleotide repeatexpansion comprises at least 30 GGGGCC repeats, more than 30 GGGGCCrepeats, more than 100 GGGGCC repeats, more than 500 GGGGCC repeats, ormore than 1000 GGGGCC repeats. In certain embodiments, thehexanucleotide repeat expansion is associated with nuclear foci. Incertain embodiments, C9ORF72 associated RAN translation products areassociated with nuclear foci. In certain embodiments, the C9ORF72associated RAN translation products are are poly-(glycine-proline),poly-(glycine-alanine), and poly-(glycine-arginine). In certainembodiments, the compositions and methods described herein are usefulfor reducing C9ORF72 mRNA levels, C9ORF72 protein levels, C9ORF72 RANtranslation products, and nuclear foci. In certain embodiments, thecompositions and methods described herein are useful for selectivelyreducing C9ORF72 pathogenic associated mRNA variants. Such reduction canoccur in a time-dependent manner or in a dose-dependent manner.

Also provided are methods useful for preventing, treating, ameliorating,and slowing progression of diseases associated with C9ORF72. In certainembodiments, such C9ORF72 associated diseases are neurodegenerativediseases. In certain embodiments, the neurodegenerative disease isamyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD),corticobasal degeneration syndrome (CBD), atypical Parkinsoniansyndrome, and olivopontocerebellar degeneration (OPCD).

Such diseases can have one or more risk factors, causes, or outcomes incommon. Certain risk factors and causes for development of aneurodegenerative disease, and, in particular, ALS and FTD, includegenetic predisposition and older age.

In certain embodiments, methods of treatment include administering aC9ORF72 antisense compound to an individual in need thereof. In certainembodiments, the antisense compound is a single-stranded modifiedoligonucleotide. In certain embodiments, the single-stranded modifiedoligonucleotide is complementary to a C9ORF72 nucleic acid.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Additionally, as used herein, the use of “and” means “and/or” unlessstated otherwise. Furthermore, the use of the term “including” as wellas other forms, such as “includes” and “included”, is not limiting.Also, terms such as “element” or “component” encompass both elements andcomponents comprising one unit and elements and components that comprisemore than one subunit, unless specifically stated otherwise.

Compounds of the invention include variations of the disclosed compoundsin which one or more hydrogen, carbon, nitrogen, oxygen, or sulfur atomsis replaced with a stable isotope of the same element.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this disclosure,including, but not limited to, patents, patent applications, publishedpatent applications, articles, books, treatises, and GENBANK AccessionNumbers and associated sequence information obtainable through databasessuch as National Center for Biotechnology Information (NCBI) and otherdata referred to throughout in the disclosure herein are herebyexpressly incorporated by reference for the portions of the documentdiscussed herein, as well as in their entirety.

DEFINITIONS

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart.

Standard techniques may be used for chemical synthesis, and chemicalanalysis.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-OCH₂CH₂—OCH₃ and MOE) refers toan O-methoxyethyl modification of the 2′ position of a furanose ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means anucleoside comprising a MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituentat the 2′-position of the furanose ring other than H or OH. In certainembodiments, 2′-substituted nucleosides include nucleosides withbicyclic sugar modifications.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“Administered concomitantly” refers to the co-administration of twopharmaceutical agents in any manner in which the pharmacological effectsof both are manifest in the patient at the same time. Concomitantadministration does not require that both pharmaceutical agents beadministered in a single pharmaceutical composition, in the same dosageform, or by the same route of administration. The effects of bothpharmaceutical agents need not manifest themselves at the same time. Theeffects need only be overlapping for a period of time and need not becoextensive.

“Administering” means providing a pharmaceutical agent to an animal, andincludes, but is not limited to administering by a medical professionaland self-administering.

“Amelioration” refers to a lessening, slowing, stopping, or reversing ofat least one indicator of the severity of a condition or disease. Theseverity of indicators may be determined by subjective or objectivemeasures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antibody” refers to a molecule characterized by reacting specificallywith an antigen in some way, where the antibody and the antigen are eachdefined in terms of the other. Antibody may refer to a complete antibodymolecule or any fragment or region thereof, such as the heavy chain, thelight chain, Fab region, and Fc region.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding. Examples of antisense compounds include single-stranded anddouble-stranded compounds, such as, antisense oligonucleotides, siRNAs,shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels inthe presence of an antisense compound complementary to a target nucleicacid compared to target nucleic acid levels or in the absence of theantisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridizationof a compound with a target nucleic acid, wherein the outcome or effectof the hybridization is either target degradation or target occupancywith concomitant stalling of the cellular machinery involving, forexample, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding segment of a target nucleic acid.

“Base complementarity” refers to the capacity for the precise basepairing of nucleobases of an antisense oligonucleotide withcorresponding nucleobases in a target nucleic acid (i.e.,hybridization), and is mediated by Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen binding between corresponding nucleobases.

“Bicyclic sugar” means a furanose ring modified by the bridging of twoatoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleoside” (also BNA) means a nucleoside having a sugarmoiety comprising a bridge connecting two carbon atoms of the sugarring, thereby forming a bicyclic ring system. In certain embodiments,the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring.

“C9ORF72 antisense transcript” means transcripts produced from thenon-coding strand (also antisense strand and template strand) of theC9ORF72 gene. The C9ORF72 antisense transcript differs from thecanonically transcribed “C9ORF72 sense transcript”, which is producedfrom the coding strand (also sense strand) of the C9ORF72 gene. Incertain embodiments, a C9ORF72 antisense transcript is SEQ ID NO: 18.

“C9ORF72 antisense transcript specific inhibitor” refers to any agentcapable of specifically inhibiting the expression of C9ORF72 antisensetranscript and/or its expression products at the molecular level. Asused herein, “specific” means reducing or inhibiting expression ofC9ORF72 antisense transcript without reducing non-target transcript toan appreciable degree (e.g., a C9ORF72 antisense transcript specificinhibitor reduces expression of C9ORF72 antisense transcript, but doesnot reduce expression of C9ORF72 sense transcript to an appreciabledegree). C9ORF72 specific antisense transcript inhibitors includeantisense compounds, siRNAs, aptamers, antibodies, peptides, smallmolecules, and other agents capable of inhibiting the expression ofC9ORF72 antisense transcript and/or its expression products, such asC9ORF72 antisense transcript associated RAN translation products.

“C9ORF72 associated disease” means any disease associated with anyC9ORF72 nucleic acid or expression product thereof. Such diseases mayinclude a neurodegenerative disease. Such neurodegenerative diseases mayinclude ALS and FTD. In certain embodiments, the C9ORF72 associateddisease is caused by (or is associated with) a hexanucleotide repeatexpansion. In certain embodiments, the hexanucleotide repeat expansionmay comprise GGGGCC, GGGGGG, GGGGGC, or GGGGCG repeated at least 30times, more than 30 times, more than 100 times, more than 500 times, ormore than 1000 times.

“C9ORF72 associated RAN translation products” means aberrant peptide ordi-peptide polymers translated through RAN translation (i.e.,repeat-associated, and non-ATG-dependent translation). In certainembodiments, the C9ORF72 associated RAN translation products are any ofpoly-(glycine-proline), poly-(glycine-alanine), andpoly-(glycine-arginine).

“C9ORF72 nucleic acid” means any nucleic acid encoding C9ORF72. Forexample, in certain embodiments, a C9ORF72 nucleic acid includes a DNAsequence encoding C9ORF72, an RNA sequence transcribed from DNA encodingC9ORF72 including genomic DNA comprising introns and exons (i.e.,pre-mRNA), and an mRNA sequence encoding C9ORF72. “C9ORF72 mRNA” meansan mRNA encoding a C9ORF72 protein.

“C9ORF72 pathogenic associated mRNA variant” means the C9ORF72 mRNAvariant processed from a C9ORF72 pre-mRNA variant containing thehexanucleotide repeat. A C9ORF72 pre-mRNA contains the hexanucleotiderepeat when transcription of the pre-mRNA begins in the region from thestart site of exon 1A to the start site of exon 1B, e.g., nucleotides1107 to 1520 of the genomic sequence (SEQ ID NO: 2, the complement ofGENBANK Accession No. NT_008413.18 truncated from nucleosides 27535000to 27565000). In certain embodiments, the level of a C9ORF72 pathogenicassociated mRNA variant is measured to determine the level of a C9ORF72pre-mRNA containing the hexanucleotide repeat in a sample.

“C9ORF72 specific inhibitor” refers to any agent capable of specificallyinhibiting the expression of C9ORF72 mRNA and/or C9ORF72 protein at themolecular level. For example, C9ORF72 specific inhibitors includenucleic acids (including antisense compounds), siRNAs, aptamers,antibodies, peptides, small molecules, and other agents capable ofinhibiting the expression of C9ORF72 mRNA and/or C9ORF72 protein.Similarly, in certain embodiments, C9ORF72 specific inhibitors mayaffect other molecular processes in an animal.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“cEt” or “constrained ethyl” means a bicyclic nucleoside having a sugarmoiety comprising a bridge connecting the 4′-carbon and the 2′-carbon,wherein the bridge has the formula: 4′-CH(CH₃)—O-2′.

“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleosidecomprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleosides is chemically distinct from a region having nucleosideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions, each position having a pluralityof subunits.

“Co-administration” means administration of two or more pharmaceuticalagents to an individual. The two or more pharmaceutical agents may be ina single pharmaceutical composition, or may be in separatepharmaceutical compositions. Each of the two or more pharmaceuticalagents may be administered through the same or different routes ofadministration. Co-administration encompasses parallel or sequentialadministration.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid.

“Comprise,” “comprises,” and “comprising” will be understood to implythe inclusion of a stated step or element or group of steps or elementsbut not the exclusion of any other step or element or group of steps orelements.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Designing” or“designed to” refer to the process of designing anoligomeric compound that specifically hybridizes with a selected nucleicacid molecule.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, in drugs that are injected, the diluent may be aliquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose may be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections may be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses may be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” in the context of modulating an activity or oftreating or preventing a condition means the administration of thatamount of pharmaceutical agent to a subject in need of such modulation,treatment, or prophylaxis, either in a single dose or as part of aseries, that is effective for modulation of that effect, or fortreatment or prophylaxis or improvement of that condition. The effectiveamount may vary among individuals depending on the health and physicalcondition of the individual to be treated, the taxonomic group of theindividuals to be treated, the formulation of the composition,assessment of the individual's medical condition, and other relevantfactors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. Such structures include, but are not limited to the products oftranscription and translation.

“Focus” or “foci” means a nuclear or cytoplasmic body comprising aC9ORF72 transcript. In certain embodiments, a focus comprises at leastone C9ORF72 transcript. In certain embodiments, C9ORF72 foci comprisetranscripts comprising any of the following hexanucleotide repeats:GGGGCC, GGGGGG, GGGGGC, and/or GGGGCG.

“Fully complementary” or “100% complementary” means each nucleobase of afirst nucleic acid has a complementary nucleobase in a second nucleicacid. In certain embodiments, a first nucleic acid is an antisensecompound and a target nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as a “gap” and theexternal regions may be referred to as the “wings.”

“Hexanucleotide repeat expansion” means a series of six bases (forexample, GGGGCC, GGGGGG, GGGGCG, or GGGGGC) repeated at least twice. Incertain embodiments, the hexanucleotide repeat expansion may be locatedin intron 1 of a C9ORF72 nucleic acid. In certain embodiments, apathogenic hexanucleotide repeat expansion includes at least 30, morethan 30, more than 100, more than 500, or more than 1000 repeats ofGGGGCC, GGGGGG, GGGGCG, or GGGGGC in a C9ORF72 nucleic acid and isassociated with disease. In certain embodiments, the repeats areconsecutive. In certain embodiments, the repeats are interrupted by 1 ormore nucleobases. In certain embodiments, a wild-type hexanucleotiderepeat expansion includes 23 or fewer repeats of GGGGCC, GGGGGG, GGGGCG,or GGGGGC in a C9ORF72 nucleic acid. In certain embodiments, the repeatsare consecutive. In certain embodiments, the repeats are interrupted by1 or more nucleobases.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude, but are not limited to, an antisense compound and a targetnucleic acid. In certain embodiments, complementary nucleic acidmolecules include, but are not limited to, an antisense oligonucleotideand a nucleic acid target.

“Identifying an animal having a C9ORF72 associated disease” meansidentifying an animal having been diagnosed with a C9ORF72 associateddisease or predisposed to develop a C9ORF72 associated disease.Individuals predisposed to develop a C9ORF72 associated disease includethose having one or more risk factors for developing a C9ORF72associated disease, including, having a personal or family history orgenetic predisposition of one or more C9ORF72 associated diseases. Suchidentification may be accomplished by any method including evaluating anindividual's medical history and standard clinical tests or assessments,such as genetic testing.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Inhibiting C9ORF72” means reducing the level or expression of a C9ORF72mRNA and/or protein. In certain embodiments, C9ORF72 mRNA and/or proteinlevels are inhibited in the presence of an antisense compound targetingC9ORF72, including an antisense oligonucleotide targeting C9ORF72, ascompared to expression of C9ORF72 mRNA and/or protein levels in theabsence of a C9ORF72 antisense compound, such as an antisenseoligonucleotide.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity and does not necessarily indicatea total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Linked nucleosides” means adjacent nucleosides linked together by aninternucleoside linkage.

“Locked nucleic acid” or “LNA” or “LNA nucleosides” means nucleic acidmonomers having a bridge connecting two carbon atoms between the 4′ and2′-position of the nucleoside sugar unit, thereby forming a bicyclicsugar. Examples of such bicyclic sugar include, but are not limited toA) α-L-Methyleneoxy (4′-CH₂—O-2′) LNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) LNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) LNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH₂—N(R)—O-2′) LNA, asdepicted below.

As used herein, LNA compounds include, but are not limited to, compoundshaving at least one bridge between the 4′ and the 2′ position of thesugar wherein each of the bridges independently comprises 1 or from 2 to4 linked groups independently selected from —[C(R₁)(R₂)]_(n)—,—C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—, —C(═S)—, —O—, —Si(R₁)₂—,—S(═O), and —N(R₁)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; eachR₁ and R₂ is, independently, H, a protecting group, hydroxyl, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, a heterocycle radical, a substitutedheterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclicradical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,N₃, COOJ₁, acyl(C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl or a protecting group.

Examples of 4′-2′ bridging groups encompassed within the definition ofLNA include, but are not limited to one of formulae: —[C(R₁)(R₂)]_(n)—,—[C(R₁)(R₂)]_(n)—O—, —C(R₁R₂)—N(R₁)—O— or —C(R₁R₂)—O—N(R₁)—.Furthermore, other bridging groups encompassed with the definition ofLNA are 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′,4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R₁)-2′ and 4′-CH₂—N(R₁)—O-2′- bridges,wherein each R₁ and R₂ is, independently, H, a protecting group orC₁-C₁₂ alkyl.

Also included within the definition of LNA according to the inventionare LNAs in which the 2′-hydroxyl group of the ribosyl sugar ring isconnected to the 4′ carbon atom of the sugar ring, thereby forming amethyleneoxy (4′-CH₂—O-2′) bridge to form the bicyclic sugar moiety. Thebridge can also be a methylene —CH₂—) group connecting the 2′ oxygenatom and the 4′ carbon atom, for which the term methyleneoxy(4′-CH₂—O-2′) LNA is used. Furthermore; in the case of the bicylic sugarmoiety having an ethylene bridging group in this position, the termethyleneoxy (4′-CH₂CH₂—O-2′) LNA is used. α-L-methyleneoxy(4′-CH₂—O-2′), an isomer of methyleneoxy (4′-CH₂—O-2′) LNA is alsoencompassed within the definition of LNA, as used herein.

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e., aphosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine,guanine, thymidine, or uracil. An “unmodified nucleobase” means thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C), and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, amodified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, and/or modifiednucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified internucleoside linkage, modified sugar, and/or modifiednucleobase.

“Modified sugar” means substitution and/or any change from a naturalsugar moiety.

“Monomer” means a single unit of an oligomer. Monomers include, but arenot limited to, nucleosides and nucleotides, whether naturally occurringor modified.

“Motif” means the pattern of unmodified and modified nucleoside in anantisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA(2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Non-complementary nucleobase” refers to a pair of nucleobases that donot form hydrogen bonds with one another or otherwise supporthybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes, but is not limited to, ribonucleic acids (RNA),deoxyribonucleic acids (DNA), single-stranded nucleic acids,double-stranded nucleic acids, small interfering ribonucleic acids(siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable ofbase pairing with another nucleobase. For example, in DNA, adenine (A)is complementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase refers to a nucleobase of an antisense compound that iscapable of base pairing with a nucleobase of its target nucleic acid.For example, if a nucleobase at a certain position of an antisensecompound is capable of hydrogen bonding with a nucleobase at a certainposition of a target nucleic acid, then the position of hydrogen bondingbetween the oligonucleotide and the target nucleic acid is considered tobe complementary at that nucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound such as for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo, or tricyclo sugar mimetics, e.g., non furanose sugar units.Nucleotide mimetic includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage). Sugar surrogate overlaps with the slightly broader termnucleoside mimetic but is intended to indicate replacement of the sugarunit (furanose ring) only. The tetrahydropyranyl rings provided hereinare illustrative of an example of a sugar surrogate wherein the furanosesugar group has been replaced with a tetrahydropyranyl ring system.“Mimetic” refers to groups that are substituted for a sugar, anucleobase, and/or internucleoside linkage. Generally, a mimetic is usedin place of the sugar or sugar-internucleoside linkage combination, andthe nucleobase is maintained for hybridization to a selected target.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Off-target effect” refers to an unwanted or deleterious biologicaleffect associated with modulation of RNA or protein expression of a geneother than the intended target nucleic acid.

“Oligomeric compound” or “oligomer” means a polymer of linked monomericsubunits which is capable of hybridizing to at least a region of anucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection(e.g., bolus injection) or infusion. Parenteral administration includessubcutaneous administration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g., intrathecal orintracerebroventricular administration.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Without limitation, as used herein, peptide refers topolypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeuticbenefit when administered to an individual. For example, in certainembodiments, an antisense oligonucleotide targeted to C9ORF72 is apharmaceutical agent.

“Pharmaceutically acceptable derivative” encompasses pharmaceuticallyacceptable salts, conjugates, prodrugs or isomers of the compoundsdescribed herein.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked)nucleobases of a nucleic acid. In certain embodiments, a portion is adefined number of contiguous nucleobases of a target nucleic acid. Incertain embodiments, a portion is a defined number of contiguousnucleobases of an antisense compound.

“Prevent” or “preventing” refers to delaying or forestalling the onsetor development of a disease, disorder, or condition for a period of timefrom minutes to days, weeks to months, or indefinitely.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form within the body or cells thereof bythe action of endogenous enzymes or other chemicals or conditions.

“Prophylactically effective amount” refers to an amount of apharmaceutical agent that provides a prophylactic or preventativebenefit to an animal.

“Region” is defined as a portion of the target nucleic acid having atleast one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ positionof the sugar portion of the nucleotide. Ribonucleotides may be modifiedwith any of a variety of substituents.

“Salts” mean a physiologically and pharmaceutically acceptable salts ofantisense compounds, i.e., salts that retain the desired biologicalactivity of the parent oligonucleotide and do not impart undesiredtoxicological effects thereto.

“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid.

“Shortened” or “truncated” versions of antisense oligonucleotides taughtherein have one, two or more nucleosides deleted.

“Side effects” means physiological responses attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, renal function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand. A “single-stranded modifiedoligonucleotide” means a modified oligonucleotide which is nothybridized to a complementary strand.

“Sites,” as used herein, are defined as unique nucleobase positionswithin a target nucleic acid.

“Slows progression” means decrease in the development of the disease.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity between an antisenseoligonucleotide and a target nucleic acid to induce a desired effect,while exhibiting minimal or no effects on non-target nucleic acids underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays and therapeutictreatments.

“Stringent hybridization conditions” or “stringent conditions” refer toconditions under which an oligomeric compound will hybridize to itstarget sequence, but to a minimal number of other sequences.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Target” refers to a protein, the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” and“nucleic acid target” all mean a nucleic acid capable of being targetedby antisense compounds.

“Target region” means a portion of a target nucleic acid to which one ormore antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to an individual.

“Treat” or “treating” or “treatment” means administering a compositionto effect an alteration or improvement of a disease or condition.

“Unmodified nucleobases” means the purine bases adenine (A) and guanine(G), and the pyrimidine bases (T), cytosine (C), and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturallyoccuring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

“Wing segment” means a plurality of nucleosides modified to impart to anoligonucleotide properties such as enhanced inhibitory activity,increased binding affinity for a target nucleic acid, or resistance todegradation by in vivo nucleases.

Certain Embodiments

Certain embodiments provide compositions and methods for reducing totalC9ORF72 mRNA and protein expression.

Certain embodiments provide compositions and methods for reducingC9ORF72 pathogenic associated mRNA variants.

Certain embodiments provide methods for the treatment, prevention,amelioration, or slowing progression of diseases associated with C9ORF72in an individual in need thereof. Also contemplated are methods for thepreparation of a medicament for the treatment, prevention, oramelioration of a disease associated with C9ORF72. C9ORF72 associateddiseases include neurodegenerative diseases. In certain embodiments, theneurodegenerative disease may be ALS or FTD. In certain embodiments, theneurodegenerative disease may be familial or sporadic.

The present disclosure provided the following non-limiting numberedembodiments:

Embodiment 1

A compound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence comprising at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, or at least 20 consecutive nucleobases of any of the nucleobasesequences of SEQ ID NOs: 22-55.

Embodiment 2

The compound of embodiment 1, wherein the nucleobase sequence of themodified oligonucleotide is at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% complementary to SEQ ID NO: 1 or SEQ IDNO: 2.

Embodiment 3

The compound of embodiments 1 and 2, wherein the modifiedoligonucleotide is a single-stranded modified oligonucleotide.

Embodiment 4

The compound of any of embodiments 1-3, wherein the modifiedoligonucleotide comprises at least one modified internucleoside linkage.

Embodiment 5

The compound of embodiment 4, wherein the at least one modifiedinternucleoside linkage is a phosphorothioate internucleoside linkage.

Embodiment 6

The compound of embodiments 4 and 5, wherein the modifiedoligonucleotide comprises at least one phosphodiester linkage.

Embodiment 7

The compound of embodiment 5, wherein each modified internucleosidelinkage is a phosphorothioate internucleoside linkage.

Embodiment 8

The compound of any of embodiments 1-7, wherein at least one nucleosidecomprises a modified nucleobase.

Embodiment 9

The compound of embodiment 8, wherein the modified nucleobase is a5-methylcytosine.

Embodiment 10

The compound of any of embodiments 1-9, wherein at least one nucleosideof the modified oligonucleotide comprises a modified sugar.

Embodiment 11

The compound of embodiment 10, wherein each nucleoside of the modifiedoligonucleotide comprises a modified sugar.

Embodiment 12

The compound of embodiments 10 or 11, wherein the at least one modifiedsugar is a bicyclic sugar.

Embodiment 13

The compound of embodiment 12, wherein the bicyclic sugar comprises achemical bridge between the 4′ and 2′ positions of the sugar, whereinthe chemical bridge is selected from: 4′-CH(R)—O-2′ and 4′-(CH₂)₂—O-2′,wherein R is independently H, C₁-C₆ alkyl, and C₁-C₆ alkoxy.

Embodiment 14

The compound of embodiment 13, wherein the chemical bridge is4′-CH(R)—O-2′ and wherein R is methyl.

Embodiment 15

The compound of embodiment 13, wherein the chemical bridge is4′-CH(R)—O-2′ and wherein R is H.

Embodiment 16

The compound of embodiment 13, wherein the chemical bridge is4′-CH(R)—O-2′ and wherein R is —CH₂—O—CH₃.

Embodiment 17

The compound of embodiments 10 or 11, wherein the at least one modifiedsugar comprises a 2′-O-methoxyethyl group.

Embodiment 18

The compound of any of embodiments 1-10 or 12-16, wherein the modifiedoligonucleotide is a gapmer.

Embodiment 19

The compound of embodiment 18, wherein the gapmer is any of a 3-8-7 MOEgapmer, a 3-10-7 MOE gapmer, a 4-8-6 MOE gapmer, a 4-10-6 MOE gapmer, a6-10-4 MOE gapmer, a 6-8-4 MOE gapmer, a 7-8-3 MOE gapmer, or a 7-10-3MOE gapmer.

Embodiment 20

The compound of claim 5, wherein the modified oligonucleotide comprisesinternucleoside linkages in any of the following patterns:soossssssssssooooss, sooossssssssssoooss, sooooossssssssssoss,soooooossssssssssss, soooosssssssssooss, sooosssssssssoooss,sooooosssssssssoss, soosssssssssoooss, soooosssssssssoss,sosssssssssooooss, or sooooosssssssssss, wherein,

-   -   s=a phosphorothioate linkage, and    -   o=a phosphodiester linkage.

Embodiment 19

A composition comprising the compound of any preceding embodiment orsalt thereof and at least one of a pharmaceutically acceptable carrieror diluent.

Embodiment 20

The composition of embodiment 19, further comprising a C9ORF72 antisensetranscript specific inhibitor.

Embodiment 21

The composition of embodiment 20, wherein the C9ORF72 antisensetranscript specific inhibitor is an antisense compound.

Embodiment 22

The composition of embodiment 21, wherein the antisense compound is amodified oligonucleotide.

Embodiment 23

The composition of embodiment 22, wherein the modified oligonucleotideis single-stranded.

Embodiment 24

The composition of embodiments 22 or 23, wherein the modifiedoligonucleotide has a nucleobase sequence that is at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% complementary to a C9ORF72 antisense transcript.

Embodiment 25

The composition of embodiment 24, wherein the C9ORF72 antisensetranscript has the nucleobase sequence of SEQ ID NO: 18.

Embodiment 26

A method comprising administering to an animal the compound orcomposition of any preceding embodiment.

Embodiment 27

The method of embodiment 26, wherein the animal is a human.

Embodiment 28

The method of embodiments 26 and 27, wherein administering the compoundprevents, treats, ameliorates, or slows progression of a C9ORF72associated disease.

Embodiment 29

The method of embodiment 28, wherein the C9ORF72 associated disease iscaused by a hexanucleotide repeat expansion.

Embodiment 30

The method of embodiment 28, wherein the C9ORF72 associated disease isamyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD),corticobasal degeneration syndrome (CBD), atypical Parkinsoniansyndrome, and olivopontocerebellar degeneration (OPCD).

Embodiment 31

The method of embodiments 26-30, wherein the administering reducesnuclear foci.

Embodiment 32

The method of embodiments 26-31, wherein the administering reducesexpression of C9ORF72 associated RAN translation products.

Embodiment 33

The method of embodiment 32, wherein the C9ORF72 associated RANtranslation products are any of poly-(glycine-proline),poly-(glycine-alanine), and poly-(glycine-arginine).

Embodiment 34

Use of the compound or composition of any of embodiments 1-33 for themanufacture of a medicament for treating a neurodegenerative disorder.

Embodiment 35

A compound consisting of a modified oligonucleotide according to thefollowing formula, or a salt thereof:

Embodiment 36

A composition consisting of the sodium salt of a modifiedoligonucleotide according to the following formula:

Embodiment 37

A compound consisting of a modified oligonucleotide according to thefollowing formula, or a salt thereof:

Embodiment 38

A composition consisting of the sodium salt of a modifiedoligonucleotide according to the following formula:

Embodiment 39

A compound consisting of a modified oligonucleotide according to thefollowing formula, or a salt thereof:

Embodiment 40

A composition consisting of the sodium salt of a modifiedoligonucleotide according to the following formula:

Embodiment 41

A compound consisting of a modified oligonucleotide according to thefollowing formula, or a salt thereof:

Embodiment 42

A composition consisting of the sodium salt of a modifiedoligonucleotide according to the following formula:

Embodiment 43

The compound or composition of any of embodiments 1-25 or 35-42, whereinthe compound or composition is capable of reducing human C9ORF72 mRNA orprotein expression in a mammal.

Embodiment 44

The compound or composition of any of embodiments 1-25 or 35-42, whereinthe compound or composition is capable of ameliorating at least onesymptom of amyotrophic lateral sclerosis (ALS), frontotemporal dementia(FTD), corticobasal degeneration syndrome (CBD), atypical Parkinsoniansyndrome, or olivopontocerebellar degeneration (OPCD).

Embodiment 45

The compound or composition of embodiment 44, wherein the symptom of ALSis any of motor deficit, anxiety, and denervation.

Embodiment 46

The compound or composition of any of embodiments 1-25 or 35-42, whereinthe compound or composition is capable of delaying progression ofdisease.

Embodiment 47

The compound or composition of any of embodiments 1-25 or 35-42, whereinthe compound or composition is capable of extending survival.

Embodiment 48

The compound or composition of any of embodiments 1-25 or 35-42, whereinthe compound or composition is capable of reducing C9ORF72 associatedRAN translation products.

Embodiment 49

The compound or composition of any of embodiments 1-25 or 35-42, whereinthe compound or composition is capable of selectively reducing C9ORF72pathogenic associated mRNA variants.

Embodiment 50

The compound or composition of any of embodiments 1-25 or 35-42, whereinthe compound or composition is capable of reducing nuclear foci.

Embodiment 51

A compound consisting of a modified oligonucleotide according to thefollowing formula, or a salt thereof:

Embodiment 52

A method comprising administering to an animal the compound ofembodiment 51.

Embodiment 53

The method of embodiment 52, wherein the animal is a human.

Embodiment 54

The method of embodiment 53, wherein the administering inhibits C9ORF72.

Embodiment 55

The method of embodiment 53, wherein the administering prevents, treats,ameliorates, or slows progression of a C9ORF72 associated disease.

Embodiment 56

The method of embodiment 55, wherein the C9ORF72 associated disease iscaused by a hexanucleotide repeat expansion.

Embodiment 57

The method of embodiment 55, wherein the C9ORF72 associated disease isany of amyotrophic lateral sclerosis (ALS), frontotemporal dementia(FTD), corticobasal degeneration syndrome (CBD), atypical Parkinsoniansyndrome, or olivopontocerebellar degeneration (OPCD).

Embodiment 58

The method of embodiment 53, wherein the administering reduces nuclearfoci.

Embodiment 59

The method of embodiment 53, wherein the administering reducesexpression of C9ORF72 associated RAN translation products.

Embodiment 60

The method of embodiment 59, wherein the C9ORF72 associated RANtranslation products are any of poly-(glycine-proline),poly-(glycine-alanine), and poly-(glycine-arginine).

Embodiment 61

A composition consisting of the sodium salt of a modifiedoligonucleotide according to the following formula:

Embodiment 62

A method comprising administering to an animal the composition ofembodiment 60.

Embodiment 63

The method of embodiment 62, wherein the animal is a human.

Embodiment 64

The method of embodiment 63, wherein the administering inhibits C9ORF72.

Embodiment 65

The method of embodiment 62, wherein the administering prevents, treats,ameliorates, or slows progression of a C9ORF72 associated disease.

Embodiment 66

The method of embodiment 65, wherein the C9ORF72 associated disease iscaused by a hexanucleotide repeat expansion.

Embodiment 67

The method of embodiment 65, wherein the C9ORF72 associated disease isany of amyotrophic lateral sclerosis (ALS), frontotemporal dementia(FTD), corticobasal degeneration syndrome (CBD), atypical Parkinsoniansyndrome, or olivopontocerebellar degeneration (OPCD).

Embodiment 68

The method of embodiment 63, wherein the administering reduces nuclearfoci.

Embodiment 69

The method of embodiment 63, wherein the administering reducesexpression of C9ORF72 associated RAN translation products.

Embodiment 70

The method of embodiment 69, wherein the C9ORF72 associated RANtranslation products are any of poly-(glycine-proline),poly-(glycine-alanine), and poly-(glycine-arginine).

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound may be “antisense” to a target nucleic acid, meaningthat is is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a C9ORF72nucleic acid is 12 to 30 subunits in length. In other words, suchantisense compounds are from 12 to 30 linked subunits. In certainembodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18to 24, 19 to 22, or 20 linked subunits. In certain embodiments, theantisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a rangedefined by any two of the above values. In some embodiments theantisense compound is an antisense oligonucleotide, and the linkedsubunits are nucleosides.

In certain embodiments antisense oligonucleotides targeted to a C9ORF72nucleic acid may be shortened or truncated. For example, a singlesubunit may be deleted from the 5′ end (5′ truncation), or alternativelyfrom the 3′ end (3′ truncation). A shortened or truncated antisensecompound targeted to a C9ORF72 nucleic acid may have two subunitsdeleted from the 5′ end, or alternatively may have two subunits deletedfrom the 3′ end, of the antisense compound. Alternatively, the deletednucleosides may be dispersed throughout the antisense compound, forexample, in an antisense compound having one nucleoside deleted from the5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisensecompound, the additional subunit may be located at the 5′ or 3′ end ofthe antisense compound. When two or more additional subunits arepresent, the added subunits may be adjacent to each other, for example,in an antisense compound having two subunits added to the 5′ end (5′addition), or alternatively to the 3′ end (3′ addition), of theantisense compound. Alternatively, the added subunits may be dispersedthroughout the antisense compound, for example, in an antisense compoundhaving one subunit added to the 5′ end and one subunit added to the 3′end.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a C9ORF72nucleic acid have chemically modified subunits arranged in patterns, ormotifs, to confer to the antisense compounds properties such as enhancedinhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound may optionally serve as a substrate for thecellular endonuclease RNase H, which cleaves the RNA strand of anRNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides that supports RNaseH cleavage is positioned betweenexternal regions having a plurality of nucleotides that are chemicallydistinct from the nucleosides of the internal region. In the case of anantisense oligonucleotide having a gapmer motif, the gap segmentgenerally serves as the substrate for endonuclease cleavage, while thewing segments comprise modified nucleosides. In certain embodiments, theregions of a gapmer are differentiated by the types of sugar moietiescomprising each distinct region. The types of sugar moieties that areused to differentiate the regions of a gapmer may in some embodimentsinclude β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modifiednucleosides (such 2′-modified nucleosides may include 2′-MOE, and2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (suchbicyclic sugar modified nucleosides may include those having a4′-(CH₂)n-O-2′ bridge, where n=1 or n=2 and 4′-CH₂—O—CH₂-2′).Preferably, each distinct region comprises uniform sugar moieties. Thewing-gap-wing motif is frequently described as “X-Y-Z”, where “X”represents the length of the 5′ wing region, “Y” represents the lengthof the gap region, and “Z” represents the length of the 3′ wing region.As used herein, a gapmer described as “X-Y-Z” has a configuration suchthat the gap segment is positioned immediately adjacent to each of the5′ wing segment and the 3′ wing segment. Thus, no interveningnucleotides exist between the 5′ wing segment and gap segment, or thegap segment and the 3′ wing segment. Any of the antisense compoundsdescribed herein can have a gapmer motif. In some embodiments, X and Zare the same, in other embodiments they are different. In a preferredembodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30 or more nucleotides. Thus, gapmers described herein include, butare not limited to, for example 5-10-5, 5-10-4, 4-10-4, 4-10-3, 3-10-3,2-10-2, 5-9-5, 5-9-4, 4-9-5, 5-8-5, 5-8- 4, 4-8-5, 5-7-5, 4-7-5, 5-7-4,or 4-7-4.

In certain embodiments, the antisense compound has a “wingmer” motif,having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Zconfiguration as described above for the gapmer configuration. Thus,wingmer configurations described herein include, but are not limited to,for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10,8-2, 2-13, 5-13, 5-8, or 6-8.

In certain embodiments, antisense compounds targeted to a C9ORF72nucleic acid possess a 5-10-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a C9ORF72nucleic acid possess a 5-8-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a C9ORF72nucleic acid possess sugar modifications in any of the followingpatterns: eeekkdddddddkkeee, eekkddddddddkkeee, ekddddddddekekeee,kekeddddddddekeke, and ekekddddddddkekee; wherein,

-   -   e=a 2′-O-methoxyethyl modified nucleoside    -   d=a 2′-deoxynucleoside, and    -   k=a cEt nucleoside.

In certain embodiments, an antisense compound targeted to a C9ORF72nucleic acid has a gap-narrowed motif. In certain embodiments, agap-narrowed antisense oligonucleotide targeted to a C9ORF72 nucleicacid has a gap segment of 9, 8, 7, or 6 2′-deoxynucleotides positionedimmediately adjacent to and between wing segments of 5, 4, 3, 2, or 1chemically modified nucleosides. In certain embodiments, the chemicalmodification comprises a bicyclic sugar. In certain embodiments, thebicyclic sugar comprises a 4′ to 2′ bridge selected from among:4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or 2; and 4′-CH₂—O—CH₂-2′. Incertain embodiments, the bicyclic sugar is comprises a 4′-CH(CH₃)—O-2′bridge. In certain embodiments, the chemical modification comprises anon-bicyclic 2′-modified sugar moiety. In certain embodiments, thenon-bicyclic 2′-modified sugar moiety comprises a 2′-O-methylethyl groupor a 2′-O-methyl group.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode C9ORF72 include, without limitation,the following: the complement of GENBANK Accession No. NM_001256054.1(incorporated herein as SEQ ID NO: 1), the complement of GENBANKAccession No. NT_008413.18 truncated from nucleobase 27535000 to27565000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No.BQ068108.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No.NM_018325.3 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No.DN993522.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No.NM_145005.5 (incorporated herein as SEQ ID NO: 6), GENBANK Accession No.DB079375.1 (incorporated herein as SEQ ID NO: 7), GENBANK Accession No.BU194591.1 (incorporated herein as SEQ ID NO: 8), Sequence Identifier4141_014_A (incorporated herein as SEQ ID NO: 9), and SequenceIdentifier 4008_73_A (incorporated herein as SEQ ID NO: 10), and GENBANKAccession No. NW 001101662.1 truncated from nucleosides 8522000 to U.S.Pat. No. 8,552,000 (incorporated herein as SEQ ID NO: 11).

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region may encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor C9ORF72 can be obtained by accession number from sequence databasessuch as NCBI and such information is incorporated herein by reference.In certain embodiments, a target region may encompass the sequence froma 5′ target site of one target segment within the target region to a 3′target site of another target segment within the same target region.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceeding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment may specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin a target region. In certain embodiments, reductions in C9ORF72mRNA levels are indicative of inhibition of C9ORF72 expression.Reductions in levels of a C9ORF72 protein are also indicative ofinhibition of target mRNA expression. Reduction in the presence ofexpanded C9ORF72 RNA foci are indicative of inhibition of C9ORF72expression. Further, phenotypic changes are indicative of inhibition ofC9ORF72 expression. For example, improved motor function and respirationmay be indicative of inhibition of C9ORF72 expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a C9ORF72 nucleic acid. The most common mechanismof hybridization involves hydrogen bonding (e.g., Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In certainembodiments, the antisense compounds provided herein are specificallyhybridizable with a C9ORF72 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as a C9ORF72 nucleicacid).

Non-complementary nucleobases between an antisense compound and aC9ORF72 nucleic acid may be tolerated provided that the antisensecompound remains able to specifically hybridize to a target nucleicacid. Moreover, an antisense compound may hybridize over one or moresegments of a C9ORF72 nucleic acid such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to a C9ORF72 nucleic acid, a target region, targetsegment, or specified portion thereof. Percent complementarity of anantisense compound with a target nucleic acid can be determined usingroutine methods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e., 100%complementary) to a target nucleic acid, or specified portion thereof.For example, an antisense compound may be fully complementary to aC9ORF72 nucleic acid, or a target region, or a target segment or targetsequence thereof. As used herein, “fully complementary” means eachnucleobase of an antisense compound is capable of precise base pairingwith the corresponding nucleobases of a target nucleic acid. Forexample, a 20 nucleobase antisense compound is fully complementary to atarget sequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound may or may not be fully complementary tothe target sequence, depending on whether the remaining 10 nucleobasesof the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases may be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they may be contiguous (i.e., linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no morethan 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a C9ORF72 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a C9ORF72 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 9 nucleobase portion of a target segment. Incertain embodiments, the antisense compounds are complementary to atleast a 10 nucleobase portion of a target segment. In certainembodiments, the antisense compounds, are complementary to at least an11 nucleobase portion of a target segment. In certain embodiments, theantisense compounds, are complementary to at least a 12 nucleobaseportion of a target segment. In certain embodiments, the antisensecompounds, are complementary to at least a 13 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds, arecomplementary to at least a 14 nucleobase portion of a target segment.In certain embodiments, the antisense compounds, are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least a 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a targetsegment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number, or portion thereof. As usedherein, an antisense compound is identical to the sequence disclosedherein if it has the same nucleobase pairing ability. For example, a RNAwhich contains uracil in place of thymidine in a disclosed DNA sequencewould be considered identical to the DNA sequence since both uracil andthymidine pair with adenine. Shortened and lengthened versions of theantisense compounds described herein as well as compounds havingnon-identical bases relative to the antisense compounds provided hereinalso are contemplated. The non-identical bases may be adjacent to eachother or dispersed throughout the antisense compound. Percent identityof an antisense compound is calculated according to the number of basesthat have identical base pairing relative to the sequence to which it isbeing compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is comparedto an equal length portion of the target nucleic acid. In certainembodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleobase portion is compared to an equal lengthportion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide iscompared to an equal length portion of the target nucleic acid. Incertain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equallength portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to a C9ORF72nucleic acid comprise one or more modified internucleoside linkages. Incertain embodiments, the modified internucleoside linkages areinterspersed throughout the antisense compound. In certain embodiments,the modified internucleoside linkages are phosphorothioate linkages. Incertain embodiments, each internucleoside linkage of an antisensecompound is a phosphorothioate internucleoside linkage. In certainembodiments, the antisense compounds targeted to a C9ORF72 nucleic acidcomprise at least one phosphodiester linkage and at least onephosphorothioate linkage.

In certain embodiments, antisense compounds targeted to a C9ORF72nucleic acid possess internucleoside linkages in any of the followingpatterns: soooossssssssssooss, sooosssssssssooss, soosssssssssooss, andsosssssssssoooss; wherein,

-   -   s=a phosphorothioate linkage, and    -   o=a phosphodiester linkage.        Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprisechemically modified ribofuranose ring moieties. Examples of chemicallymodified ribofuranose rings include without limitation, addition ofsubstitutent groups (including 5′ and 2′ substituent groups, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂are each independently H, C₁-C₁₂ alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′-F-5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂₀CH₃ substituentgroups. The substituent at the 2′ position can also be selected fromallyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F,O(CH₂)₂SCH₃, O(CH₂)₂—O—N((R_(m))(R_(n)), O—CH₂—C(═O)—N((R_(m))(R_(n)),and O—CH₂—C(═O)—N(R₁)—(CH₂)₂—N((R_(m)))(R_(n)), where each R₁, (R_(m))and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleic acids(BNAs) include without limitation nucleosides comprising a bridgebetween the 4′ and the 2′ ribosyl ring atoms. In certain embodiments,antisense compounds provided herein include one or more BNA nucleosideswherein the bridge comprises one of the formulas: 4′-(CH₂)—O-2′ (LNA);4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ and4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845,issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof seePCT/US2008/068922 published as WO/2009/006478, published Jan. 8, 2009);4′-CH₂—N(OCH₃)-2′ (and analogs thereof see PCT/US2008/064591 publishedas WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (seepublished U.S. Patent Application US2004-0171570, published Sep. 2,2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₀ alkyl, or a protectinggroup (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008);4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya et al., J. Org. Chem., 2009, 74,118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof seePCT/US2008/066154 published as WO 2008/154401, published on Dec. 8,2008).

Further bicyclic nucleosides have been reported in published literature(see for example: Srivastava et al., J. Am. Chem. Soc., 2007, 129(26)8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372;Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braaschet al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol.Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 5633-5638; Singh et al., Chem. Commun., 1998, 4,455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org.Chem., 1998, 63, 10035-10039; U.S. Pat. Nos. 7,399,845; 7,053,207;7,034,133; 6,794,499; 6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S.Patent Publication Nos.: US2008-0039618; US2007-0287831; US2004-0171570;U.S. patent application Ser. No. 12/129,154; 61/099,844; 61/097,787;61/086,231; 61/056,564; 61/026,998; 61/026,995; 60/989,574;International applications WO 2007/134181; WO 2005/021570; WO2004/106356; and PCT International Applications Nos.: PCT/US2008/068922;PCT/US2008/066154; and PCT/US2008/064591). Each of the foregoingbicyclic nucleosides can be prepared having one or more stereochemicalsugar configurations including for example α-L-ribofuranose andβ-D-ribofuranose (see PCT international application PCT/DK98/00393,published on Mar. 25, 1999 as WO 99/14226).

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ carbon atoms of the pentofuranosyl sugarmoiety including without limitation, bridges comprising 1 or from 1 to 4linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein: x is 0, 1, or 2; nis 1, 2, 3, or 4; each R_(a) and R_(b) is, independently, H, aprotecting group, hydroxyl, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycleradical, substituted heterocycle radical, heteroaryl, substitutedheteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclicradical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H),substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₀ alkyl, substituted C₁-C₁₀alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₀ aminoalkyl, substituted C₁-C₁₀ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′,4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′-wherein each R is, independently, H, a protecting group or C₁-C₁₀ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a4′-(CH₂)—O-2′ bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4′to 2′ bridge wherein such bridges include without limitation,α-L-4′-(CH₂)—O-2′, β-D-4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′,4′-CH₂—N(R)—O-2′, 4′-CH(CH₃)—O-2′, 4′-CH₂—S-2′, 4′-CH₂—N(R)-2′,4′-CH₂—CH(CH₃)-2′, and 4′-(CH₂)₃-2′, wherein R is H, a protecting groupor C₁-C₁₂ alkyl.

In certain embodiment, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₀ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thiol.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₀ alkoxy, substituted C₁-C₁₀ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₀ alkyl orsubstituted C₁-C₁₀ alkyl.

The synthesis and preparation of adenine, cytosine, guanine,5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a4′-CH₂—O-2′ bridge, along with their oligomerization, and nucleic acidrecognition properties have been described (Koshkin et al., Tetrahedron,1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has alsobeen described in WO 98/39352 and WO99/14226.

Analogs of various bicyclic nucleosides that have 4′ to 2′ bridginggroups such as 4′-CH₂—O-2′ and 4′-CH₂—S-2′, have also been prepared(Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of oligodeoxyribonucleotide duplexes comprising bicyclicnucleosides for use as substrates for nucleic acid polymerases has alsobeen described (Wengel et al., WO 99/14226). Furthermore, synthesis of2′-amino-BNA, a novel conformationally restricted high-affinityoligonucleotide analog has been described in the art (Singh et al., J.Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and2′-methylamino-BNA's have been prepared and the thermal stability oftheir duplexes with complementary RNA and DNA strands has beenpreviously reported.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Frier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy(4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F)methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to asconstrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H)methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic(4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA,and (K) vinyl BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protectinggroup, C₁-C₆ alkyl or C₁-C₆ alkoxy.

As used herein, the term “modified tetrahydropyran nucleoside” or“modified THP nucleoside” means a nucleoside having a six-memberedtetrahydropyran “sugar” substituted for the pentofuranosyl residue innormal nucleosides and can be referred to as a sugar surrogate. ModifiedTHP nucleosides include, but are not limited to, what is referred to inthe art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA),manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10,841-854) or fluoro HNA (F-HNA) having a tetrahydropyranyl ring system asillustrated below.

In certain embodiment, sugar surrogates are selected having the formula:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an oligomeric compound oroligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protectinggroup, a linked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen,substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁,J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isother than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄,q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following formula:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′,2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129 (26), 8362-8379).

In certain embodiments, antisense compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horváth et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al. Nucleosides, Nucleotides & Nucleic Acids, 2005,24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenylnucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the cyclohexenyl nucleoside analog to an antisense compound orone of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an antisense compound and the otherof T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugarsubstituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known inthe art and are suitable as sugar surrogates that can be used to modifynucleosides for incorporation into oligomeric compounds as providedherein (see for example review article: Leumann, Christian J. Bioorg. &Med. Chem., 2002, 10, 841-854). Such ring systems can undergo variousadditional substitutions to further enhance their activity.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F,O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, wheren and m are from 1 to about 10. Other 2′-substituent groups can also beselected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving pharmacokinetic properties, or a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In certain embodiments, modifiednucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem.,1997, 272, 11944-12000). Such 2′-MOE substitution have been described ashaving improved binding affinity compared to unmodified nucleosides andto other modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504;Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,1997, 16, 917-926).

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′ substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N((R_(m))(R_(n)), orO—CH₂—C(═O)—N((R_(m))(R_(n)), where each (R_(m)) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃”, “2′-O-methyl” or “2′-methoxy”each refers to a nucleoside comprising a sugar comprising an —OCH₃ groupat the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

Methods for the preparations of modified sugars are well known to thoseskilled in the art. Some representative U.S. patents that teach thepreparation of such modified sugars include without limitation, U.S.:4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633;5,700,920; 5,792,847 and 6,600,032 and International ApplicationPCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 onDec. 22, 2005, and each of which is herein incorporated by reference inits entirety.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleosides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleosides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a bicyclic nucleoside having a(4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the(4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wingsof a gapmer motif.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceuticallyacceptable active or inert substances for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

An antisense compound targeted to a C9ORF72 nucleic acid can be utilizedin pharmaceutical compositions by combining the antisense compound witha suitable pharmaceutically acceptable diluent or carrier. Apharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in one embodiment, employed in the methodsdescribed herein is a pharmaceutical composition comprising an antisensecompound targeted to a C9ORF72 nucleic acid and a pharmaceuticallyacceptable diluent. In certain embodiments, the pharmaceuticallyacceptable diluent is PBS. In certain embodiments, the antisensecompound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acid from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof C9ORF72 nucleic acids can be tested in vitro in a variety of celltypes. Cell types used for such analyses are available from commercialvendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio,Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville,Md.) and are cultured according to the vendor's instructions usingcommercially available reagents (e.g. Invitrogen Life Technologies,Carlsbad, Calif.). Illustrative cell types include, but are not limitedto, HepG2 cells, Hep3B cells, and primary hepatocytes.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a LIPOFECTAMINEconcentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation.

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein. In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE. Antisense oligonucleotides are used at higherconcentrations ranging from 625 to 20,000 nM when transfected usingelectroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOLReagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a C9ORF72 nucleic acid can beassayed in a variety of ways known in the art. For example, targetnucleic acid levels can be quantitated by, e.g., Northern blot analysis,competitive polymerase chain reaction (PCR), or quantitative real-timePCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Quantitative real-time PCR can beconveniently accomplished using the commercially available ABI PRISM7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence DetectionSystem (PE-Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. Methods of quantitative real-time PCR arewell known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000instrument (PE Applied Biosystems) is used to measure RIBOGREENfluorescence.

Probes and primers are designed to hybridize to a C9ORF72 nucleic acid.Methods for designing real-time PCR probes and primers are well known inthe art, and may include the use of software such as PRIMER EXPRESSSoftware (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of C9ORF72 nucleic acids can be assessed bymeasuring C9ORF72 protein levels. Protein levels of C9ORF72 can beevaluated or quantitated in a variety of ways well known in the art,such as immunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS). Antibodies directed to a target can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art. Antibodies useful for the detection of mouse,rat, monkey, and human C9ORF72 are commercially available.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of C9ORF72 andproduce phenotypic changes, such as, improved motor function andrespiration. In certain embodiments, motor function is measured byrotarod, grip strength, pole climb, open field performance, balancebeam, hindpaw footprint testing in the animal. In certain embodiments,respiration is measured by whole body plethysmograph, invasiveresistance, and compliance measurements in the animal. Testing may beperformed in normal animals, or in experimental disease models. Foradministration to animals, antisense oligonucleotides are formulated ina pharmaceutically acceptable diluent, such as phosphate-buffered saline(PBS) or artificial cerebrospinal fluid (aCSF). Administration includesparenteral routes of administration, such as intraperitoneal,intravenous, and subcutaneous, as well as central routes ofadministration such as intracerebroventricular or intrathecal.Calculation of antisense oligonucleotide dosage and dosing frequency iswithin the abilities of those skilled in the art, and depends uponfactors such as route of administration and animal body weight.Following a period of treatment with antisense oligonucleotides, RNA isisolated from CNS tissue or CSF and changes in C9ORF72 nucleic acidexpression are measured.

Targeting C9ORF72

Antisense oligonucleotides described herein may hybridize to a C9ORF72nucleic acid in any stage of RNA processing. For example, describedherein are antisense oligonucleotides that are complementary to apre-mRNA or a mature mRNA. Additionally, antisense oligonucleotidesdescribed herein may hybridize to any element of a C9ORF72 nucleic acid.For example, described herein are antisense oligonucleotides that arecomplementary to an exon, an intron, the 5′ UTR, the 3′ UTR, a repeatregion, a hexanucleotide repeat expansion, a splice junction, anexon:exon splice junction, an exonic splicing silencer (ESS), an exonicsplicing enhancer (ESE), exon 1a, exon 1b, exon 1c, exon 1d, exon 1e,exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10,exon11, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6,intron 7, intron 8, intron 9, or intron 10 of a C9ORF72 nucleic acid.

In certain embodiments, antisense oligonucleotides described hereinhybridize to all variants of C9ORF72. In certain embodiments, theantisense oligonucleotides described herein selectively hybridize tocertain variants of C9ORF72. In certain embodiments, the antisenseoligonucleotides described herein selectively hybridize to variants ofC9ORF72 containing a hexanucleotide repeat expansion. In certainembodiments, the antisense oligonucleotides described herein selectivelyhybridize to pre-mRNA variants containing the hexanucleotide repeat. Incertain embodiments, pre-mRNA variants of C9ORF72 containing ahexanucleotide repeat expansion include SEQ ID NO: 1-3 and 6-10. Incertain embodiments, such hexanucleotide repeat expansion comprises atleast 24 repeats of any of GGGGCC, GGGGGG, GGGGGC, or GGGGCG.

In certain embodiments, the antisense oligonucleotides described hereininhibit expression of all variants of C9ORF72. In certain embodiments,the antisense oligonucleotides described herein inhibit expression ofall variants of C9ORF72 equally. In certain embodiments, the antisenseoligonucleotides described herein preferentially inhibit expression ofone or more variants of C9ORF72. In certain embodiments, the antisenseoligonucleotides described herein preferentially inhibit expression ofvariants of C9ORF72 containing a hexanucleotide repeat expansion. Incertain embodiments, the antisense oligonucleotides described hereinselectively inhibit expression of pre-mRNA variants containing thehexanucleotide repeat. In certain embodiments, the antisenseoligonucleotides described herein selectively inhibit expression ofC9ORF72 pathogenic associated mRNA variants. In certain embodiments,pre-mRNA variants of C9ORF72 containing a hexanucleotide repeatexpansion include SEQ ID NO: 1-3 and 6-10. In certain embodiments, suchhexanucleotide repeat expansion comprises at least 24 repeats of any ofGGGGCC, GGGGGG, GGGGGC, or GGGGCG. In certain embodiments, thehexanucleotide repeat expansion forms nuclear foci. In certainembodiments, antisense oligonucleotides described herein are useful forreducing nuclear foci. Nuclear foci may be reduced in terms of percentof cells with foci as well as number of foci per cell.

Selective Inhibition of Certain Pathogenic Associated Variants

In certain examples herein, primer probe set RTS3905 detects an mRNAvariant (e.g. NM_001256054.1) processed from a pre-mRNA variantcontaining the hexanucleotide repeat. The mRNA variant processed from apre-mRNA variant containing the hexanucleotide repeat (i.e., the“C9ORF72 pathogenic associated mRNA variant”). A pre-mRNA contains thehexanucleotide repeat when transcription of the pre-mRNA begins in theregion from the start site of exon 1A to the start site of exon 1B,e.g., nucleotides 1107 to 1520 of the genomic sequence (SEQ ID NO: 2,the complement of GENBANK Accession No. NT_008413.18 truncated fromnucleosides 27535000 to 27565000). Oligonucleotides were designed inthis region to selectively target the pre-mRNA variant containing thehexanucleotide repeat. RTS3905 measures an mRNA product (i.e. theC9ORF72 pathogenic associated mRNA variant) of the pre-mRNA variantcontaining the hexanucleotide repeat and, therefore, measures thereduction of the pre-mRNA variant containing the hexanucleotide repeat.

C9ORF72 Features

Antisense oligonucleotides described herein may hybridize to any C9ORF72variant at any state of processing within any element of the C9ORF72gene. For example, antisense oligonucleotides described herein mayhybridize to an exon, an intron, the 5′ UTR, the 3′ UTR, a repeatregion, a hexanucleotide repeat expansion, a splice junction, anexon:exon splice junction, an exonic splicing silencer (ESS), an exonicsplicing enhancer (ESE), exon 1a, exon 1b, exon 1c, exon 1d, exon 1e,exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10,exon 11, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6,intron 7, intron 8, intron 9, or intron 10. For example, antisenseoligonucleotides may target any of the exons characterized below inTables 1-5 for the various C9ORF72 variants described below. Antisenseoligonucleotides described herein may also target variants notcharacterized below and such variants are characterized in GENBANK.Moreover, antisense oligonucleotides described herein may also targetelements other than exons and such elements are characterized inGENBANK.

TABLE 1 Functional Segments for NM_001256054.1 (SEQ ID NO: 1) Start Stopsite in site in mRNA mRNA reference reference Exon start stop to SEQ toSEQ Number site site ID NO: 2 ID NO: 2 exon 1C 1 158 1137 1294 exon 2159 646 7839 8326 exon 3 647 706 9413 9472 exon 4 707 802 12527 12622exon 5 803 867 13354 13418 exon 6 868 940 14704 14776 exon 7 941 105716396 16512 exon 8 1058 1293 18207 18442 exon 9 1294 1351 24296 24353exon 10 1352 1461 26337 26446 exon 11 1462 3339 26581 28458

TABLE 2 Functional Segments for NM_018325.3 (SEQ ID NO: 4) Start Stopsite in site in mRNA mRNA reference reference Exon start stop to SEQ toSEQ Number site site ID NO: 2 ID NO: 2 exon 1B 1 63 1510 1572 exon 2 64551 7839 8326 exon 3 552 611 9413 9472 exon 4 612 707 12527 12622 exon 5708 772 13354 13418 exon 6 773 845 14704 14776 exon 7 846 962 1639616512 exon 8 963 1198 18207 18442 exon 9 1199 1256 24296 24353 exon 101257 1366 26337 26446 exon 11 1367 3244 26581 28458

TABLE 3 Functional Segments for NM_145005.5 (SEQ ID NO: 6) Start Stopsite in site in mRNA mRNA reference reference start stop to SEQ to SEQExon Number site site ID NO: 2 ID NO: 2 exon 1A 1 80 1137 1216 exon 2 81568 7839 8326 exon 3 569 628 9413 9472 exon 4 629 724 12527 12622 exon5B (exon 5 725 1871 13354 14500 into intron 5)

TABLE 4 Functional Segments for DB079375.1 (SEQ ID NO: 7) Start Stopsite in site in mRNA mRNA reference reference start stop to SEQ to SEQExon Number site site ID NO: 2 ID NO: 2 exon 1E 1 35 1135 1169 exon 2 36524 7839 8326 exon 3 (EST ends 525 562 9413 9450 before end of fullexon)

TABLE 5 Functional Segments for BU194591.1 (SEQ ID NO: 8) Start Stopsite in site in mRNA mRNA reference reference start stop to SEQ to SEQExon Number site site ID NO: 2 ID NO: 2 exon 1D 1 36 1241 1279 exon 2 37524 7839 8326 exon 3 525 584 9413 9472 exon 4 585 680 12527 12622 exon5B (exon 5 681 798 13354 13465 into intron 5)Certain Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions described herein. In certain embodiments, the individualhas a neurodegenerative disease. In certain embodiments, the individualis at risk for developing a neurodegenerative disease, including, butnot limited to, ALS or FTD. In certain embodiments, the individual hasbeen identified as having a C9ORF72 associated disease. In certainembodiments, provided herein are methods for prophylactically reducingC9ORF72 expression in an individual. Certain embodiments includetreating an individual in need thereof by administering to an individuala therapeutically effective amount of an antisense compound targeted toa C9ORF72 nucleic acid.

In one embodiment, administration of a therapeutically effective amountof an antisense compound targeted to a C9ORF72 nucleic acid isaccompanied by monitoring of C9ORF72 levels in an individual, todetermine an individual's response to administration of the antisensecompound. An individual's response to administration of the antisensecompound may be used by a physician to determine the amount and durationof therapeutic intervention.

In certain embodiments, administration of an antisense compound targetedto a C9ORF72 nucleic acid results in reduction of C9ORF72 expression byat least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 99%, or a range defined by any two of these values. In certainembodiments, administration of an antisense compound targeted to aC9ORF72 nucleic acid results in improved motor function and respirationin an animal. In certain embodiments, administration of a C9ORF72antisense compound improves motor function and respiration by at least15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or99%, or a range defined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to C9ORF72 are used for the preparation of amedicament for treating a patient suffering or susceptible to aneurodegenerative disease including ALS and FTD.

Certain Combination Therapies

In certain embodiments, one or more pharmaceutical compositionsdescribed herein are co-administered with one or more otherpharmaceutical agents. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat the same disease, disorder,or condition as the one or more pharmaceutical compositions describedherein. In certain embodiments, such one or more other pharmaceuticalagents are designed to treat a different disease, disorder, or conditionas the one or more pharmaceutical compositions described herein. Incertain embodiments, such one or more other pharmaceutical agents aredesigned to treat an undesired side effect of one or more pharmaceuticalcompositions described herein. In certain embodiments, one or morepharmaceutical compositions described herein are co-administered withanother pharmaceutical agent to treat an undesired effect of that otherpharmaceutical agent. In certain embodiments, one or more pharmaceuticalcompositions described herein are co-administered with anotherpharmaceutical agent to produce a combinational effect. In certainembodiments, one or more pharmaceutical compositions described hereinare co-administered with another pharmaceutical agent to produce asynergistic effect.

In certain embodiments, one or more pharmaceutical compositionsdescribed herein and one or more other pharmaceutical agents areadministered at the same time. In certain embodiments, one or morepharmaceutical compositions described herein and one or more otherpharmaceutical agents are administered at different times. In certainembodiments, one or more pharmaceutical compositions described hereinand one or more other pharmaceutical agents are prepared together in asingle formulation. In certain embodiments, one or more pharmaceuticalcompositions described herein and one or more other pharmaceuticalagents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition described hereininclude Riluzole (Rilutek), Lioresal (Lioresal), and Dexpramipexole.

In certain embodiments, pharmaceutical agents that may beco-administered with a C9ORF72 specific inhibitor described hereininclude, but are not limited to, an additional C9ORF72 inhibitor.

In certain embodiments, pharmaceutical agents that may beco-administered with a C9ORF72 specific inhibitor described hereininclude, but are not limited to, a C9ORF72 antisense transcript specificinhibitor. In certain embodiments, the C9ORF72 antisense transcriptspecific inhibitor is an antisense compound. In certain embodiments, theantisense compound is a modified oligonucleotide. In certainembodiments, the modified oligonucleotide is single-stranded.

In certain embodiments, the co-administered pharmaceutical agent isadministered prior to administration of a pharmaceutical compositiondescribed herein. In certain embodiments, the co-administeredpharmaceutical agent is administered following administration of apharmaceutical composition described herein. In certain embodiments theco-administered pharmaceutical agent is administered at the same time asa pharmaceutical composition described herein. In certain embodimentsthe dose of a co-administered pharmaceutical agent is the same as thedose that would be administered if the co-administered pharmaceuticalagent was administered alone. In certain embodiments the dose of aco-administered pharmaceutical agent is lower than the dose that wouldbe administered if the co-administered pharmaceutical agent wasadministered alone. In certain embodiments the dose of a co-administeredpharmaceutical agent is greater than the dose that would be administeredif the co-administered pharmaceutical agent was administered alone.

In certain embodiments, the co-administration of a second compoundenhances the effect of a first compound, such that co-administration ofthe compounds results in an effect that is greater than the effect ofadministering the first compound alone. In other embodiments, theco-administration results in effects that are additive of the effects ofthe compounds when administered alone. In certain embodiments, theco-administration results in effects that are supra-additive of theeffects of the compounds when administered alone. In certainembodiments, the first compound is an antisense compound. In certainembodiments, the second compound is an antisense compound.

Certain Comparator Compositions

In certain embodiments, compounds described herein are more tolerablethan ISIS 576816, ISIS 576974, ISIS 577061, ISIS 577065, and/or ISIS577083. ISIS 576816, ISIS 576974, ISIS 577061, ISIS 577065, and ISIS577083 were selected as comparator compounds because they exhibited highlevels of dose-dependent reduction of C9ORF72 mRNA in various studiesdescribed in WO2014/062691. Thus, ISIS 576816, ISIS 576974, ISIS 577061,ISIS 577065, and ISIS 577083 were deemed highly efficacious and potentcompounds. Additionally, ISIS 577065, ISIS 577056, and ISIS 576816described in WO2014/062691 are structurally similar as compoundsdescribed herein. For example, ISIS 577065 has a 16 nucleobase overlapwith ISIS 801287; ISIS 577056 has a 16 nucleobase overlap with ISIS806679; ISIS 576816 has an 18 nucleobase overlap with ISIS 802473(18-mer); and ISIS 576816 has an 18 nucleobase overlap with ISIS 802459.

In certain embodiments, ISIS 576816, a 5-10-5 MOE gapmer, having asequence of (from 5′ to 3′) GCCTTACTCTAGGACCAAGA (incorporated herein asSEQ ID NO: 21), wherein each internucleoside linkage is aphosphorothioate linkage, each cytosine is a 5-methylcytosine, and eachof nucleosides 1-5 and 16-20 comprise a 2′-O-methoxyethyl group, whichwas previously described in WO2014/062691, incorporated herein byreference, is a comparator compound. ISIS 576816 achieved an average FOBscore of 7.00 in a study of acute tolerability in mice (see Example 3hereinbelow). Certain compounds described herein achieved a lower FOBscore in a similar study of acute tolerability in mice (see Example 2hereinbelow), including ISIS 801287, ISIS 806679, ISIS 802473, and ISIS802459. Therefore, certain compounds described herein are more tolerablethan comparator compound ISIS 576816.

In certain embodiments, ISIS 576974, a 5-10-5 MOE gapmer, having asequence of (from 5′ to 3′) GGGACACTACAAGGTAGTAT (incorporated herein asSEQ ID NO: 56), wherein each internucleoside linkage is aphosphorothioate linkage, each cytosine is a 5-methylcytosine, and eachof nucleosides 1-5 and 16-20 comprise a 2′-O-methoxyethyl group, whichwas previously described in WO2014/062691, incorporated herein byreference, is a comparator compound. ISIS 576974 achieved an average FOBscore of 5.67 in a study of acute tolerability in mice (see Example 3hereinbelow). Certain compounds described herein achieved a lower FOBscore in a similar study of acute tolerability in mice (see Example 2hereinbelow), including ISIS 801287, ISIS 806679, ISIS 802473, and ISIS802459. Therefore, certain compounds described herein are more tolerablethan comparator compound ISIS 576974.

In certain embodiments, ISIS 577061, a 5-10-5 MOE gapmer, having asequence of (from 5′ to 3′) TACAGGCTGCGGTTGTTTCC (incorporated herein asSEQ ID NO: 57), wherein each internucleoside linkage is aphosphorothioate linkage, each cytosine is a 5-methylcytosine, and eachof nucleosides 1-5 and 16-20 comprise a 2′-O-methoxyethyl group, whichwas previously described in WO2014/062691, incorporated herein byreference, is a comparator compound. ISIS 577061 achieved an average FOBscore of 7.00 in a study of acute tolerability in mice (see Example 3hereinbelow). Certain compounds described herein achieved a lower FOBscore in a similar study of acute tolerability in mice (see Example 2hereinbelow), including ISIS 801287, ISIS 806679, ISIS 802473, and ISIS802459. Therefore, certain compounds described herein are more tolerablethan comparator compound ISIS 577061.

In certain embodiments, ISIS 577065, a 5-10-5 MOE gapmer, having asequence of (from 5′ to 3′) CCCGGCCCCTAGCGCGCGAC (incorporated herein asSEQ ID NO: 58), wherein each internucleoside linkage is aphosphorothioate linkage, each cytosine is a 5-methylcytosine, and eachof nucleosides 1-5 and 16-20 comprise a 2′-O-methoxyethyl group, whichwas previously described in WO2014/062691, incorporated herein byreference, is a comparator compound. ISIS 577065 achieved an average FOBscore of 6.00 in a study of acute tolerability in mice (see Example 3hereinbelow). Certain compounds described herein achieved a lower FOBscore in a similar study of acute tolerability in mice (see Example 2hereinbelow), including ISIS 801287, ISIS 806679, ISIS 802473, and ISIS802459. Therefore, certain compounds described herein are more tolerablethan comparator compound ISIS 577065.

In certain embodiments, ISIS 577083, a 5-10-5 MOE gapmer, having asequence of (from 5′ to 3′) GGTAACTTCAAACTCTTGGG (incorporated herein asSEQ ID NO: 59), wherein each internucleoside linkage is aphosphorothioate linkage, each cytosine is a 5-methylcytosine, and eachof nucleosides 1-5 and 16-20 comprise a 2′-O-methoxyethyl group, whichwas previously described in WO2014/062691, incorporated herein byreference, is a comparator compound. ISIS 577083 achieved an average FOBscore of 7.00 in a study of acute tolerability in mice (see Example 3hereinbelow). Certain compounds described herein achieved a lower FOBscore in a similar study of acute tolerability in mice (see Example 2hereinbelow), including ISIS 801287, ISIS 806679, ISIS 802473, and ISIS802459. Therefore, certain compounds described herein are more tolerablethan comparator compound ISIS 577083.

In certain embodiments, ISIS 577056, a 5-10-5 MOE gapmer, having asequence of (from 5′ to 3′) AATCTTTATCAGGTCTTTTC (incorporated herein asSEQ ID NO: 60), wherein each internucleoside linkage is aphosphorothioate linkage, each cytosine is a 5-methylcytosine, and eachof nucleosides 1-5 and 16-20 comprise a 2′-O-methoxyethyl group, whichwas previously described in WO2014/062691, incorporated herein byreference, is a comparator compound. ISIS 577056 achieved an average FOBscore of 6.5 in a study of acute tolerability in mice (see Example 3hereinbelow). Certain compounds described herein achieved a lower FOBscore in a similar study of acute tolerability in mice (see Example 2hereinbelow), including ISIS 801287, ISIS 806679, ISIS 802473, and ISIS802459. Therefore, certain compounds described herein are more tolerablethan comparator compound ISIS 577056.

Certain Human Therapeutics

The human C9ORF72 antisense oligonucleotides described herein are humantherapeutics. Various parameters of potency, efficacy, and/ortolerability are being examined. Such parameters include in vitroinhibition of total C9ORF72 RNA expression, in vitro inhibition ofC9ORF72 pathogenic associated RNA variant expression, in vitro doseresponse (IC50), in vivo inhibition of total or pathogenic RNA and/orprotein in a transgenic animal containing a human C9ORF72 transgene inrelevant tissues (e.g., brain and/or spinal cord), tolerability inmouse, tolerability in rat, and/or tolerability in a primate.Tolerability markers that may be measured include blood and serumchemistry parameters, CSF chemistry parameters, body and organ weights,general observations and/or behavioral tests, and/or biochemical markerssuch as GFAP and/or AIF1. Acute or long term tolerability may bemeasured.

Certain Compositions

1. ISIS 801287

In certain embodiments, ISIS 801287 is characterized as a 4-8-6 MOEgapmer, having a sequence of (from 5′ to 3′) GCCCCTAGCGCGCGACTC(incorporated herein as SEQ ID NO: 33), wherein each of nucleosides 1-4and 13-18 are 2′-O-methoxyethylribose modified nucleosides, and each ofnucleosides 5-12 are 2′-deoxynucleosides, wherein the internucleosidelinkages between nucleosides 2 to 3, 3 to 4, 13 to 14, 14 to 15, and 15to 16 are phosphodiester linkages and the internucleoside linkagesbetween nucleosides 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to10, 10 to 11, 11 to 12, 12 to 13, 16 to 17, and 17 to 18 arephosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 801287 is described by the followingchemical notation: Ges mCeo mCeo mCes mCds Tds Ads Gds mCds Gds mCds GdsmCeo Geo Aeo mCes Tes mCe;

wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.        In certain embodiments, ISIS 801287 is described by the        following chemical structure, or a salt thereof:

Structure 1. ISIS 801287

In certain embodiments, the sodium salt of ISIS 801287 is described bythe following chemical structure:

Structure 2. The Sodium Salt of ISIS 8012872. ISIS 806679

In certain embodiments, ISIS 806679 is characterized as a 6-10-4 MOEgapmer, having a sequence of (from 5′ to 3′) GGTTAATCTTTATCAGGTCT(incorporated herein as SEQ ID NO: 49), wherein each of nucleosides 1-6and 17-20 are 2′-O-methoxyethylribose modified nucleosides, and each ofnucleosides 7-16 are 2′-deoxynucleosides, wherein the internucleosidelinkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, and17 to 18 are phosphodiester linkages and the internucleoside linkagesbetween nucleosides 1 to 2, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12,12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 18 to 19, and 19 to 20are phosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 806679 is described by the followingchemical notation: Ges Geo Teo Teo Aeo Aeo Tds mCds Tds Tds Tds Ads TdsmCds Ads Gds Geo Tes mCes Te; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 806679 is described by the followingchemical structure, or a salt thereof:

Structure 3. ISIS 806679

In certain embodiments, the sodium salt of ISIS 806679 is described bythe following chemical structure:

Structure 4. The Sodium Salt of ISIS 8066793. ISIS 802473

In certain embodiments, ISIS 802473 is characterized as a 4-8-6 MOEgapmer, having a sequence of (from 5′ to 3′) GCCTTACTCTAGGACCAA(incorporated herein as SEQ ID NO: 47), wherein each of nucleosides 1-4and 13-18 are 2′-O-methoxyethylribose modified nucleosides, and each ofnucleosides 5-12 are 2′-deoxynucleosides, wherein the internucleosidelinkages between nucleosides 2 to 3, 3 to 4, 13 to 14, 14 to 15, and 15to 16 are phosphodiester linkages and the internucleoside linkagesbetween nucleosides are 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9,9 to 10, 10 to 11, 11 to 12, 12 to 13, 16 to 17, and 17 to 18 arephosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 802473 is described by the followingchemical notation: Ges mCeo mCeo Tes Tds Ads mCds Tds mCds Tds Ads GdsGeo Aeo mCeo mCes Aes Ae; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 802473 is described by the followingchemical structure, or a salt thereof:

Structure 5. ISIS 802473

In certain embodiments, the sodium salt of ISIS 802473 is described bythe following chemical structure:

Structure 6. The Sodium Salt of ISIS 8024734. ISIS 802459

In certain embodiments, ISIS 802459 is characterized as a 3-10-7 MOEgapmer, having a sequence of (from 5′ to 3′) GCCTTACTCTAGGACCAAGA(incorporated herein as SEQ ID NO: 21), wherein each of nucleosides 1-3and 14-20 are 2′-O-methoxyethylribose modified nucleosides, and each ofnucleosides 4-13 are 2′-deoxynucleosides, wherein the internucleosidelinkages between nucleosides 2 to 3, 3 to 4, 14 to 15, 15 to 16, 16 to17, and 17 to 18 are phosphodiester linkages and the internucleosidelinkages between nucleosides are 1 to 2, 4 to 5, 5 to 6, 6 to 7, 7 to 8,8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 18 to 19, and19 to 20 are phosphorothioate linkages, and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, ISIS 802459 is described by the followingchemical notation: Ges mCeo mCeo Tds Tds Ads mCds Tds mCds Tds Ads GdsGds Aeo mCeo mCeo Aeo Aes Ges Ae; wherein,

-   -   A=an adenine,    -   mC=a 5′-methylcytosine    -   G=a guanine,    -   T=a thymine,    -   e=a 2′-O-methoxyethylribose modified sugar    -   d=a 2′-deoxyribose sugar,    -   s=a phosphorothioate internucleoside linkage, and    -   o=a phosphodiester internucleoside linkage.

In certain embodiments, ISIS 802459 is described by the followingchemical structure, or a salt thereof:

Structure 7. ISIS 802459

In certain embodiments, the sodium salt of ISIS 802459 is described bythe following chemical structure:

Structure 8. The Sodium Salt of ISIS 802459

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions, and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1 Antisense Oligonucleotides Targeting Human C9ORF72

The antisense oligonucleotides in the table below were designed as MOEgapmers. The central gap segment of each gapmer contains2′-deoxynucleosides and is flanked by wing segments on both the 5′ endand on the 3′ end containing nucleosides that each comprise a 2′-MOEgroup. The specific motif of each gapmer is listed in table below,represented by three numbers separated by hyphens. The numbers representthe number of nucleosides in the 5′-wing, the gap, and the 3′-wing,respectively. All cytosine residues throughout each oligonucleotide are5-methylcytosines. The internucleoside linkages for the gapmers aremixed phosphorothioate and phosphodiester linkages. The internucleosidelinkages for each gapmer are presented in the Linkage column, where ‘o’indicates a phosphodiester linkage and ‘s’ indicates a phosphorothioatelinkage.

Each antisense oligonucleotide listed in the table below is targeted tothe human C9ORF72 genomic sequence, designated herein as SEQ ID NO: 2(the complement of GENBANK Accession No. NT_008413.18 truncated fromnucleosides 27535000 to 27565000). “Start site” indicates the 5′-mostnucleoside to which the gapmer is targeted in the human genomicsequence. “Stop site” indicates the 3′-most nucleoside to which thegapmer is targeted human genomic sequence.

TABLE 6 Antisense oligonucleotides targeting human C9ORF72 SEQ IsisStart Stop ID No. Site Site Sequence Linkage Motif NO 791656 1445 1464CCGGCCCCTAGCGCGCGACT soossssssssssooooss 3-10-7 22 791657 1445 1464CCGGCCCCTAGCGCGCGACT sooossssssssssoooss 4-10-6 22 791658 1445 1464CCGGCCCCTAGCGCGCGACT sooooossssssssssoss 6-10-4 22 791659 1445 1464CCGGCCCCTAGCGCGCGACT soooooossssssssssss 7-10-3 22 791660 1445 1463CGGCCCCTAGCGCGCGACT soooosssssssssooss 5-9-5 23 791661 1446 1464CCGGCCCCTAGCGCGCGAC soooosssssssssooss 5-9-5 24 791662 1445 1463CGGCCCCTAGCGCGCGACT sooosssssssssoooss 4-9-6 23 791663 1446 1464CCGGCCCCTAGCGCGCGAC sooosssssssssoooss 4-9-6 24 791664 1445 1463CGGCCCCTAGCGCGCGACT sooooosssssssssoss 6-9-4 23 791665 1446 1464CCGGCCCCTAGCGCGCGAC sooooosssssssssoss 6-9-4 24 801274 1440 1459CCCTAGCGCGCGACTCCTGA sooossssssssssoooss 4-10-6 25 801275 1441 1460CCCCTAGCGCGCGACTCCTG sooossssssssssoooss 4-10-6 26 801276 1442 1461GCCCCTAGCGCGCGACTCCT sooossssssssssoooss 4-10-6 27 801277 1443 1462GGCCCCTAGCGCGCGACTCC sooossssssssssoooss 4-10-6 28 801278 1444 1463CGGCCCCTAGCGCGCGACTC sooossssssssssoooss 4-10-6 29 801279 1440 1459CCCTAGCGCGCGACTCCTGA sooooossssssssssoss 6-10-4 25 801280 1441 1460CCCCTAGCGCGCGACTCCTG sooooossssssssssoss 6-10-4 26 801281 1442 1461GCCCCTAGCGCGCGACTCCT sooooossssssssssoss 6-10-4 27 801282 1443 1462GGCCCCTAGCGCGCGACTCC sooooossssssssssoss 6-10-4 28 801283 1444 1463CGGCCCCTAGCGCGCGACTC sooooossssssssssoss 6-10-4 29 801284 1441 1458CCTAGCGCGCGACTCCTG soosssssssssoooss 4-8-6 30 801285 1442 1459CCCTAGCGCGCGACTCCT soosssssssssoooss 4-8-6 31 801286 1443 1460CCCCTAGCGCGCGACTCC soosssssssssoooss 4-8-6 32 801287 1444 1461GCCCCTAGCGCGCGACTC soosssssssssoooss 4-8-6 33 801288 1445 1462GGCCCCTAGCGCGCGACT soosssssssssoooss 4-8-6 34 801289 1446 1463CGGCCCCTAGCGCGCGAC soosssssssssoooss 4-8-6 35 801290 1441 1458CCTAGCGCGCGACTCCTG soooosssssssssoss 6-8-4 30 801291 1442 1459CCCTAGCGCGCGACTCCT soooosssssssssoss 6-8-4 31 801292 1443 1460CCCCTAGCGCGCGACTCC soooosssssssssoss 6-8-4 32 801293 1444 1461GCCCCTAGCGCGCGACTC soooosssssssssoss 6-8-4 33 801294 1445 1462GGCCCCTAGCGCGCGACT soooosssssssssoss 6-8-4 34 801295 1446 1463CGGCCCCTAGCGCGCGAC soooosssssssssoss 6-8-4 35 801296 1403 1422AGGCTGCGGTTGTTTCCCTC sooossssssssssoooss 4-10-6 36 801297 1404 1423CAGGCTGCGGTTGTTTCCCT sooossssssssssoooss 4-10-6 37 801298 1403 1421GGCTGCGGTTGTTTCCCTC soooosssssssssooss 5-9-5 38 801299 1404 1422AGGCTGCGGTTGTTTCCCT soooosssssssssooss 5-9-5 39 801300 1405 1423CAGGCTGCGGTTGTTTCCC soooosssssssssooss 5-9-5 40 801301 1403 1421GGCTGCGGTTGTTTCCCTC sooosssssssssoooss 4-9-6 38 801302 1404 1422AGGCTGCGGTTGTTTCCCT sooosssssssssoooss 4-9-6 39 801303 1405 1423CAGGCTGCGGTTGTTTCCC sooosssssssssoooss 4-9-6 40 801304 1403 1421GGCTGCGGTTGTTTCCCTC sooooosssssssssoss 6-9-4 38 801305 1404 1422AGGCTGCGGTTGTTTCCCT sooooosssssssssoss 6-9-4 39 801306 1405 1423CAGGCTGCGGTTGTTTCCC sooooosssssssssoss 6-9-4 40 801307 1403 1420GCTGCGGTTGTTTCCCTC soosssssssssoooss 4-8-6 41 801308 1404 1421GGCTGCGGTTGTTTCCCT soosssssssssoooss 4-8-6 42 801309 1405 1422AGGCTGCGGTTGTTTCCC soosssssssssoooss 4-8-6 43 801310 1406 1423CAGGCTGCGGTTGTTTCC soosssssssssoooss 4-8-6 44 801311 1403 1420GCTGCGGTTGTTTCCCTC soooosssssssssoss 6-8-4 41 801312 1404 1421GGCTGCGGTTGTTTCCCT soooosssssssssoss 6-8-4 42 801313 1405 1422AGGCTGCGGTTGTTTCCC soooosssssssssoss 6-8-4 43 801314 1406 1423CAGGCTGCGGTTGTTTCC soooosssssssssoss 6-8-4 44 801315 1403 1422AGGCTGCGGTTGTTTCCCTC sooooossssssssssoss 6-10-4 36 801316 1404 1423CAGGCTGCGGTTGTTTCCCT sooooossssssssssoss 6-10-4 37 802459 7990 8009GCCTTACTCTAGGACCAAGA soossssssssssooooss 3-10-7 21 802460 8012 8031TCTGTCTTTGGAGCCCAAAT soossssssssssooooss 3-10-7 45 802461 8186 8205CTGCGATCCCCATTCCAGTT soossssssssssooooss 3-10-7 46 802462 7990 8009GCCTTACTCTAGGACCAAGA sooossssssssssoooss 4-10-6 21 802463 8012 8031TCTGTCTTTGGAGCCCAAAT sooossssssssssoooss 4-10-6 45 802464 8186 8205CTGCGATCCCCATTCCAGTT sooossssssssssoooss 4-10-6 46 802465 7990 8009GCCTTACTCTAGGACCAAGA sooooossssssssssoss 6-10-4 21 802466 8012 8031TCTGTCTTTGGAGCCCAAAT sooooossssssssssoss 6-10-4 45 802467 8186 8205CTGCGATCCCCATTCCAGTT sooooossssssssssoss 6-10-4 46 802468 7990 8009GCCTTACTCTAGGACCAAGA soooooossssssssssss 7-10-3 21 802469 8012 8031TCTGTCTTTGGAGCCCAAAT soooooossssssssssss 7-10-3 45 802470 8186 8205CTGCGATCCCCATTCCAGTT soooooossssssssssss 7-10-3 46 802471 7992 8009GCCTTACTCTAGGACCAA sosssssssssooooss 3-8-7 47 802472 8014 8031TCTGTCTTTGGAGCCCAA sosssssssssooooss 3-8-7 48 802473 7992 8009GCCTTACTCTAGGACCAA soosssssssssoooss 4-8-6 47 802474 8014 8031TCTGTCTTTGGAGCCCAA soosssssssssoooss 4-8-6 48 802475 7992 8009GCCTTACTCTAGGACCAA soooosssssssssoss 6-8-4 47 802476 8014 8031TCTGTCTTTGGAGCCCAA soooosssssssssoss 6-8-4 48 802477 7992 8009GCCTTACTCTAGGACCAA sooooosssssssssss 7-8-3 47 802478 8014 8031TCTGTCTTTGGAGCCCAA sooooosssssssssss 7-8-3 48 806673 1370 1389GGTTAATCTTTATCAGGTCT soossssssssssooooss 3-10-7 49 806674 1371 1390TGGTTAATCTTTATCAGGTC soossssssssssooooss 3-10-7 50 806675 1372 1391CTGGTTAATCTTTATCAGGT soossssssssssooooss 3-10-7 51 806676 1370 1389GGTTAATCTTTATCAGGTCT sooossssssssssoooss 4-10-6 49 806677 1371 1390TGGTTAATCTTTATCAGGTC sooossssssssssoooss 4-10-6 50 806678 1372 1391CTGGTTAATCTTTATCAGGT sooossssssssssoooss 4-10-6 51 806679 1370 1389GGTTAATCTTTATCAGGTCT sooooossssssssssoss 6-10-4 49 806680 1371 1390TGGTTAATCTTTATCAGGTC sooooossssssssssoss 6-10-4 50 806681 1372 1391CTGGTTAATCTTTATCAGGT sooooossssssssssoss 6-10-4 51 806682 1370 1389GGTTAATCTTTATCAGGTCT soooooossssssssssss 7-10-3 49 806683 1371 1390TGGTTAATCTTTATCAGGTC soooooossssssssssss 7-10-3 50 806684 1372 1391CTGGTTAATCTTTATCAGGT soooooossssssssssss 7-10-3 51 806685 1371 1388GTTAATCTTTATCAGGTC soosssssssssoooss 4-8-6 52 806686 1372 1389GGTTAATCTTTATCAGGT soosssssssssoooss 4-8-6 53 806687 1373 1390TGGTTAATCTTTATCAGG soosssssssssoooss 4-8-6 54 806688 1440 1457CTAGCGCGCGACTCCTGA soosssssssssoooss 4-8-6 55 806689 1371 1388GTTAATCTTTATCAGGTC soooosssssssssoss 6-8-4 52 806690 1372 1389GGTTAATCTTTATCAGGT soooosssssssssoss 6-8-4 53 806691 1373 1390TGGTTAATCTTTATCAGG soooosssssssssoss 6-8-4 54 806692 1440 1457CTAGCGCGCGACTCCTGA soooosssssssssoss 6-8-4 55

Example 2 Tolerability of Antisense Oligonucleotides Targeting HumanC9ORF72 in Mice

Antisense oligonucleotides described above were tested in mice to assesstolerability of the oligonucleotides. Wild type C57/B16 mice eachreceived a single ICV dose of 700 μg of an antisense oligonucleotidelisted in the table below. Each treatment group consisted of 4 mice. At3 hours post injection, each mouse was evaluated according to 7different criteria. The 7 criteria are (1) the mouse was bright, alert,and responsive; (2) the mouse was standing or hunched without stimuli;(3) the mouse showed any movement without stimuli; (4) the mousedemonstrated forward movement after it was lifted; (5) the mousedemonstrated any movement after it was lifted; (6) the mouse respondedto a tail pinch; (7) regular breathing. For each of the 7 differentcriteria, each mouse was given a sub-score of 0 if it met the criteriaor 1 if it did not. After all of the 7 criteria were evaluated, thesub-scores were summed for each mouse and then averaged for each group.For example, if a mouse was bright, alert, and responsive 3 hours afterthe 700 ng ICV dose, and met all other criteria, it would get a summedscore of 0. If another mouse was not bright, alert, and responsive 3hours after the 700 μg ICV dose but met all other criteria, it wouldreceive a score of 1. The results are presented as the average score foreach treatment group.

TABLE 7 Acute tolerability scores Isis No. Score 791656 3.25 791657 4.25791658 3.50 791659 2.00 791660 4.00 791661 4.50 791662 5.25 791663 6.00791664 3.00 791665 5.75 801274 1.75 801275 3.75 801276 1.25 801277 1.50801278 1.75 801279 1.25 801280 6.00 801281 0.00 801282 1.25 801283 1.25801284 3.00 801285 1.00 801286 1.00 801287 1.50 801288 2.50 801289 7.00801290 6.00 801291 3.00 801292 2.00 801293 1.00 801294 3.25 801295 4.50801296 6.25 801297 4.75 801298 5.50 801299 6.25 801300 5.00 801301 6.00801302 6.50 801303 4.00 801304 5.25 801305 6.00 801306 6.00 801307 5.00801308 6.00 801309 7.00 801310 3.50 801311 5.50 801312 2.50 801313 5.25801314 4.50 801315 4.00 801316 2.00 802459 2.00 802460 6.75 802461 1.75802462 5.75 802463 6.75 802464 1.75 802465 2.25 802466 4.25 802467 0.25802468 3.25 802469 2.00 802470 0.25 802471 1.25 802472 4.00 802473 0.25802474 5.25 802475 1.00 802476 5.50 802477 2.50 802478 6.25 806673 0.00806674 0.25 806675 0.00 806676 0.00 806677 1.00 806678 0.00 806679 1.50806680 1.00 806681 0.00 806682 5.75 806683 3.75 806684 2.25 806685 1.00806686 1.00 806687 3.25 806688 3.25 806689 3.00 806690 1.25 806691 6.25

Example 3 Tolerability of Oligonucleotides from WO 2014/062691

Oligonucleotides described in WO 2014/062691 were tested in an acutetolerability study in mice. Groups of 3 wild type C57/B16 mice weretreated and analyzed as described in Example 2. The testedoligonucleotides include those listed in the table below, which are5-10-5 MOE gapmers with a full phosphorothioate backbone and eachcytosine is a 5-methylcytosine. The start and stop sites on SEQ ID NO: 2that each oligonucleotide is targeted to are shown. The results arepresented as the average score for each treatment group in the tablebelow. These results demonstrate that ISIS 576816, ISIS 576974, ISIS577061, ISIS 577065, ISIS 577083, and ISIS 577056 were poorly tolerated.

TABLE 8 Acute tolerability scores 3h aftertreatment with antisense oligo- nucleotides from WO 2014/062691 SEQ IsisStart Stop ID No. site site Sequence Score NO 576816 7990 8009GCCTTACTCTAGGACCAAGA 7.00 21 576974 28251 28270 GGGACACTACAAGGTAGTAT5.67 56 577061 1406 1425 TACAGGCTGCGGTTGTTTCC 7.00 57 577065 1446 1465CCCGGCCCCTAGCGCGCGAC 6.00 58 577083 3452 3471 GGTAACTTCAAACTCTTGGG 7.0059

TABLE 9 Acute tolerability scores 3h aftertreatment with antisense oligo- nucleotides from WO 2014/062691 SEQ IsisStart Stop ID No. site site Sequence Score NO 577056 1366 1385AATCTTTATCAGGTCTTTTC 6.5 60

Example 4 Antisense Inhibition of a Human C9ORF72 mRNA Variant in HepG2Cells

Antisense oligonucleotides described above are tested for their effectson C9ORF72 mRNA in vitro. Cultured HepG2 cells at a density of 20,000cells per well are electroporated with an antisense oligonucleotide.After a treatment period of approximately 24 hours, RNA is isolated fromthe cells and C9ORF72 mRNA levels are measured by quantitative real-timePCR. Human primer probe set RTS3905 (forward primer sequenceGGGTCTAGCAAGAGCAGGTG, designated herein as SEQ ID NO: 12; reverse primersequence GTCTTGGCAACAGCTGGAGAT, designated herein as SEQ ID NO: 13;probe sequence TGATGTCGACTCTTTGCCCACCGC, designated herein as SEQ ID NO:14—a TAQ-man primer probe set) are used. RTS3905 detects an mRNA variant(e.g. NM_001256054.1) processed from a pre-mRNA variant containing thehexanucleotide repeat. The mRNA variant processed from a pre-mRNAvariant containing the hexanucleotide repeat is herein the “C9ORF72pathogenic associated mRNA variant.” A pre-mRNA contains thehexanucleotide repeat when transcription of the pre-mRNA begins in theregion from the start site of exon 1A to the start site of exon 1B(generally nucleotides 1107 to 1520 of the genomic sequence: SEQ ID NO:2, the complement of GENBANK Accession No. NT_008413.18 truncated fromnucleosides 27535000 to 2756500. Therefore, oligonucleotides designed inthis region selectively target the pre-mRNA variant containing thehexanucleotide repeat. RTS3905 measures an mRNA product (i.e. theC9ORF72 pathogenic associated mRNA variant) of the pre-mRNA variantcontaining the hexanucleotide repeat and, therefore, measures thereduction of the pre-mRNA variant containing the hexanucleotide repeat.The levels of the C9ORF72 pathogenic associated mRNA variant arenormalized to the total RNA content of the cell, as measured byRIBOGREEN®, then the normalized mRNA variant levels are compared tothose of cells that were not treated with antisense oligonucleotide.

Example 5 Dose-Dependent Antisense Inhibition of a Human C9ORF72 mRNAVariant

Antisense oligonucleotides described above are tested at various dosesin HepG2 cells. Cells are plated at a density of 20,000 cells per welland electroporated with antisense oligonucleotide. After a treatmentperiod of approximately 16 hours or 24 hours, RNA is isolated from thecells and C9ORF72 mRNA levels are measured by quantitative real-timePCR. Human C9ORF72 primer probe set RTS3905 is used to measure theC9ORF72 pathogenic associated mRNA variant. The levels of the C9ORF72pathogenic associated mRNA variant are adjusted according to total RNAcontent, as measured by RIBOGREEN®. The half maximal inhibitoryconcentration (IC₅₀) of each oligonucleotide is calculated based on theinhibition of mRNA variant levels observed at each individual dose ofantisense oligonucleotide.

Example 6 Antisense Inhibition of C9ORF72 by Human-Rhesus Cross-ReactiveAntisense Oligonucleotides in LLC-MK2 Cells

Antisense oligonucleotides described above that are fully cross-reactivewith a rhesus C9ORF72 nucleic acid are tested for their effects onrhesus C9ORF72 mRNA in vitro. Cultured rhesus LLC-MK2 cells at a densityof 20,000 cells per well are electroporated with antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAis isolated from the cells and C9ORF72 mRNA levels are measured byquantitative real-time PCR. Primer probe set RTS3750 (forward sequenceTGTGACAGTTGGAATGCAGTGA, designated herein as SEQ ID NO: 15; reversesequence GCCACTTAAAGCAATCTCTGTCTTG, designated herein as SEQ ID NO: 16;probe sequence TCGACTCTTTGCCCACCGCCA, designated herein as SEQ ID NO:17—a TAQ-man primer probe set) is used to measure total C9ORF72 mRNAlevels. RTS3750 targets exon 2 of the mRNA transcripts and, therefore,measures total mRNA transcripts. C9ORF72 mRNA levels are adjustedaccording to total RNA content, as measured by RIBOGREEN®, then thenormalized mRNA variant levels are compared to those of cells that werenot treated with antisense oligonucleotide.

Example 7 Dose-Dependent Antisense Inhibition of Human C9ORF72 mRNA inLLC-MK2

Antisense oligonucleotides described above are tested at various dosesin LLC-MK2 cells. Cells are plated at a density of 20,000 cells per welland electroporated with antisense oligonucleotide. After a treatmentperiod of approximately 16 hours or 24 hours, RNA is isolated from thecells and C9ORF72 mRNA levels are measured by quantitative real-timePCR. Primer probe set RTS3750 is used to measure total C9ORF72 mRNAlevels. C9ORF72 mRNA levels are adjusted according to total RNA content,as measured by RIBOGREEN®, then the normalized mRNA variant levels arecompared to those of cells that were not treated with antisenseoligonucleotide.

Example 8 Antisense Inhibition of Human C9ORF72 mRNA in a TransgenicMouse Model

Antisense oligonucleotides described above are tested in two BACtransgenic mouse lines, designated herein as C9B41 and C9B183, that eachexpress a truncated human C9ORF72 gene comprising exons 1-5. Thetruncated human C9ORF72 genes of the C9B41 and C9B183 mouse linescomprise 110 and 450 hexanucleotide repeats, respectively. Each mouse ineach treatment group receives 350 μg of an antisense oligonucleotide,then expression levels of human C9ORF72 RNA are analyzed by RT-PCR. Doseresponses are also performed.

Example 9 Antisense Inhibition of C9ORF72 mRNA in Patient Fibroblasts

Antisense oligonucleotides described above were tested for their effectson C9ORF72 mRNA in vitro. The antisense oligonucleotides listed in thetable below were added to a plate before patient fibroblasts F09-152were added at a density of 20,000 cells per well. The finalconcentrations of the antisense oligonucleotides after the addition ofthe cells are listed in the table below. After 30 seconds of shaking,the cells were electroporated then transferred to a Primaria coatedculture plate. After 16 hours, RNA was isolated from the cells, andlevels of total C9ORF72 mRNA (i.e., mRNA starting from exon 1A and mRNAstarting from exon 1B) and levels of the pathogenic associated mRNA (seeExample 4) were measured by RT-qPCR. Human primer probe sets RTS 3750(see Example 6) and RTS3905 (see Example 4) were used to detect thetotal C9ORF72 mRNA and C9ORF72 pathogenic associated mRNA, respectively.The levels of the C9ORF72 total mRNA and C9ORF72 pathogenic associatedmRNA were normalized to the total RNA content of the cell, as measuredby RIBOGREEN®, then the normalized mRNA levels were compared to those ofcells that were not treated with antisense oligonucleotide. The resultsare shown in the table below. An entry of “nd” means not determined.IC₅₀ values listed as “nd” were not determined, because the target wasnot inhibited sufficiently to determine an IC₅₀. The results show thatall of the oligonucleotides listed below inhibited the pathogenicassociated C9ORF72 mRNA variant. Oligonucleotides that do notspecifically target the repeat variant pre-mRNA inhibited both thepathogenic associated C9ORF72 mRNA and total C9ORF72 mRNA.Oligonucleotides that specifically target the repeat variant pre-mRNAselectively inhibited the pathogenic associated C9ORF72 mRNA.

TABLE 10 C9ORF72 mRNA levels following antisense inhibition in patientfibroblasts Pathogenic Total associated C9ORF72 C9ORF72 Concen- mRNAmRNA tration Level IC₅₀ Level IC₅₀ Isis No. (μM) (% UTC) (μM) (% UTC)(μM) 791658 0.12 87 nd 26 <0.1 0.60 80 9 3.00 87 nd 15.00 105 4 7916640.12 85 nd 13 <0.1 0.60 101 7 3.00 125 4 15.00 114 3 801278 0.12 91 nd19 <0.1 0.60 71 7 3.00 85 nd 15.00 90 nd 801279 0.12 98 nd 57 0.12 0.6077 27 3.00 77 3 15.00 86 5 801282 0.12 100 nd 51 0.08 0.60 84 12 3.00 976 15.00 190 nd 801283 0.12 87 nd 40 <0.1 0.60 75 8 3.00 94 4 15.00 111 4801285 0.12 103 nd 35 <0.1 0.60 78 23 3.00 75 7 15.00 79 6 801286 0.1294 nd 33 <0.1 0.60 85 14 3.00 97 5 15.00 88 3 801287 0.12 85 nd 31 <0.10.60 76 11 3.00 77 9 15.00 87 2 801288 0.12 77 nd 13 <0.1 0.60 86 113.00 123 5 15.00 177 3 801292 0.12 69 nd 29 <0.1 0.60 71 12 3.00 82 315.00 71 nd 801293 0.12 77 nd 51 0.09 0.60 70 10 3.00 74 3 15.00 81 1801294 0.12 75 nd 27 <0.1 0.60 67 9 3.00 107 7 15.00 146 2 801316 0.1273 nd 35 <0.1 0.60 68 7 3.00 65 1 15.00 78 1 802459 0.12 58 0.10 75 0.180.60 15 6 3.00 2 11 15.00 1 6 802464 0.12 81 0.39 69 0.21 0.60 31 233.00 6 7 15.00 2 3 802465 0.12 40 <0.1 35 <0.1 0.60 7 7 3.00 2 nd 15.001 nd 802468 0.12 52 0.10 40 <0.1 0.60 15 13 3.00 3 7 15.00 2 nd 8024690.12 69 0.17 57 ~0.5 0.60 16 9 3.00 4 nd 15.00 1 nd 802471 0.12 71 0.2754 0.12 0.60 29 20 3.00 6 6 15.00 7 nd 802473 0.12 63 0.18 48 <0.1 0.6027 14 3.00 8 11 15.00 4 nd 802477 0.12 54 0.12 32 <0.1 0.60 16 12 3.00 411 15.00 3 3 806676 0.12 66 nd 20 <0.1 0.60 66 4 3.00 76 2 15.00 64 nd806679 0.12 71 nd 23 <0.1 0.60 68 2 3.00 78 1 15.00 84 1 806680 0.12 89nd 41 <0.1 0.60 76 13 3.00 65 7 15.00 84 nd 806690 0.12 99 nd 44 <0.10.60 88 17 3.00 85 2 15.00 77 1

Example 10 Antisense Inhibition of Human C9ORF72 mRNA in a TransgenicMouse Model

Antisense oligonucleotides described above were tested in a BACtransgenic mouse line, C9B41 (see Example 8), that expresses a humanC9ORF72 gene comprising the promoter region through exon 5 and 110hexanucleotide repeats. Each treatment group consisted of 2-3 mice. Eachmouse received a single ICVB of 350 μg of an antisense oligonucleotidelisted in the tables below or PBS. Two weeks later, the mice wereeuthanized, and expression levels of the human pathogenic associatedC9ORF72 mRNA variant and/or total human C9ORF72 mRNA were analyzed byRT-qPCR as described in Example 9. Analysis of the pathogenic associatedC9ORF72 variant mRNA levels was not completed for the oligonucleotidesthat do not specifically target the C9ORF72 repeat variant pre-mRNA. Theresults in the tables below show the average percent normalized humanC9ORF72 mRNA levels relative to the normalized average for the PBStreated group.

TABLE 11 Human C9ORF72 mRNA levels following antisense inhibition intransgenic mice Spinal Cord Cortex (% PBS treated) (% PBS treated)Pathogenic Pathogenic Isis No. variant Total variant Total 791658 6 6912 47 791659 18 72 27 57 791664 13 55 15 39 801274 49 65 27 37 801276 1047 12 29 801277 9 44 10 28 801278 8 39 10 27 801279 33 55 40 47 80128116 48 17 32 801282 18 49 31 42 801283 15 46 17 31 801285 29 51 24 36801286 37 61 37 47 801287 13 47 30 40 801288 18 52 39 48 801292 25 49 3340 801293 20 50 29 38 801310 70 80 68 70 801312 43 61 46 55 801315 39 5941 51 801316 27 56 50 58 806673 38 66 66 66 806674 78 88 98 96 806675 6786 81 85 806676 29 64 52 58 806677 61 77 69 68 806678 83 96 95 102806679 24 63 26 44 806680 29 62 41 56 806681 39 68 36 54 806684 44 69 6264 806685 69 87 56 58 806686 59 75 57 68 806687 74 88 77 80 806688 34 6242 51 806689 67 92 87 88 806690 28 61 57 68 806692 45 64 44 50

TABLE 12 Human C9ORF72 mRNA levels following antisense inhibition intransgenic mice Total human C9ORF72 mRNA (% PBS) Isis No. Spinal CordCortex 802459 28 30 802461 22 19 802464 30 28 802465 25 25 802467 13 14802468 24 26 802469 24 20 802470 18 20 802471 36 44 802473 28 38 80247515 15 802477 14 15

Example 11 Dose Dependent Antisense Inhibition of Human C9ORF72 mRNA ina Transgenic Mouse Model

Antisense oligonucleotides described above were tested in two BACtransgenic mouse lines, C9B41 and C9B183, that each express a truncatedhuman C9ORF72 gene comprising exons 1-5. The truncated human C9ORF72genes of the C9B41 and C9B183 mouse lines comprise 110 and 450hexanucleotide repeats, respectively (see Example 8). Each treatmentgroup consisted of 2-4 mice. Each mouse received a single ICVB of 30 μg,100 μg, 300 μg, or 700 μg of an antisense oligonucleotide as listed inthe tables below or PBS. Two weeks later, the mice were euthanized, andexpression levels of the human pathogenic associated C9ORF72 mRNAvariant and/or total human C9ORF72 mRNA were analyzed by RT-qPCR asdescribed in Example 9. The results in the tables below show the averagepercent normalized human C9ORF72 mRNA levels relative to the normalizedaverage for the PBS treated group. A value of 100 or greater means theantisense oligonucleotide did not reduce mRNA or increased the amount ofmRNA.

TABLE 13 Human C9ORF72 mRNA levels following dose dependnent antisenseinhibition in C9B41 transgenic mice Concen- Spinal Cord Cortex tration(% PBS treated) (% PBS treated) Isis No. (μg) Total C9 Total C9 80245930 84 85 100 63 61 300 33 26 700 29 17 802473 30 74 90 100 60 77 300 3739 700 28 26

TABLE 14 Human C9ORF72 mRNA levels following dose dependnent antisenseinhibition in C9B183 transgenic mice Concen- Spinal Cord Cortex tration(% PBS treated) (% PBS treated) Isis No. (μg) Pathogenic variantPathogenic variant 801287 30 76 10 100 37 75 300 21 32 700 11 15 80667930 72 13 100 59 100 300 21 61 700 10 16 806680 30 94 118 100 72 97 30034 89 700 30 56 806690 30 52 125 100 60 131 300 43 119 700 19 49

Example 12 Tolerability of Antisense Oligonucleotides Targeting HumanC9Orf72 in Mice

Wild type C57/B16 mice each received a single ICV dose of 700 μg of anantisense oligonucleotide listed in the table below, as described inExample 2. Each treatment group consisted of 4 mice. At 8 weekspost-injection, mice were evaluated according to 7 different criteria.The criteria are (1) the mouse was bright, alert, and responsive; (2)the mouse was standing or hunched without stimuli; (3) the mouse showedany movement without stimuli; (4) the mouse demonstrated forwardmovement after it was lifted; (5) the mouse demonstrated any movementafter it was lifted; (6) the mouse responded to tail pinching; (7)regular breathing. For each of the 7 criteria, a mouse was given asubscore of 0 if it met the criteria and 1 if it did not. After all 7criteria were evaluated, the scores were summed for each mouse andaveraged within each treatment group. The results are presented in thetable below.

Animals were sacrificed at 8 weeks. The cortex and spinal cord werecollected from each animal, and RT-PCR was performed. Expression levelsof allograft inflammatory factor (AIF1) were determined as a measure ofinflammation. Expression levels of glial fibrillary acidic protein(GFAP) were also determined as a measure of glial cell activation.Results were normalized to Gpadh and are presented relative to PBScontrol (1.0) in the table below. “N.D.” indicates there was no databecause the experiment was not performed. An asterisk indicates that thecorresponding result is the average of 1-3 mice.

TABLE 15 Tolerabilty of antisense oligonucleotides targeting C9Orf72 inmice Score 8 AIF1 GFAP SEQ ISIS weeks after (spinal AIF1 (spinal GFAP IDNo. injection cord) (cerebellum) cord) (cortex) NO. 791656 3.5 1.1* 1.3*1.0* 1.3* 22 791657 3.5 2.3* 1.0* 1.4* 0.8* 22 791658 0.0 1.9 1.2 1.41.3 22 791659 0.0 1.8 1.3 1.5 2.5 22 791660 1.8 2.5* 1.4* 1.9* 0.8* 23791661 1.8 1.7* 1.3* 1.5* 1.1* 24 791662 5.3 1.1* 1.2* 1.1* 0.8* 23791663 7.0 N.D. N.D. N.D. N.D. 24 791664 0.0 1.6 1.6 1.2 0.9 23 7916653.5 1.9* 1.5* 1.9* 0.9* 24 801274 0.0 1.2 1.3 1.3 1.1 25 801275 3.5 1.4*1.7* 1.6* 2.1* 26 801276 0.0 13.1 2.6 3.7 1.2 27 801277 0.0 2.9 1.4 2.31.5 28 801278 0.0 2.0 1.3 1.8 1.1 29 801279 0.0 1.2 1.4 1.3 1.0 25801280 5.3 1.5* 1.5* 1.4* 1.2* 26 801281 0.0 3.6 2.2 2.4 1.6 27 8012820.0 1.2 1.2 1.2 1.0 28 801283 0.0 1.4 1.4 1.0 0.9 29 801284 1.8 1.2*1.5* 0.9* 1.0* 30 801285 0.0 1.2 1.2 1.1 0.8 31 801286 0.0 1.1 1.2 1.01.1 32 801287 0.0 1.3 1.2 1.1 1.4 33 801288 0.0 1.3 1.3 1.1 0.9 34801289 7.0 N.D. N.D. N.D. N.D. 35 801290 3.5 1.2* 1.3* 1.0* 1.1* 30801291 0.0 2.2 2.2 1.5 1.1 31 801292 0.0 1.3 1.3 1.2 0.9 32 801293 0.01.6 1.6 1.3 1.5 33 801294 0.0 1.2 1.2 1.0 1.1 34 801295 1.8 1.4* 1.4*1.0* 0.9* 35 801296 7.0 N.D. N.D. N.D. N.D. 36 801297 3.5 1.0* 1.0* 0.9*0.8* 37 801307 5.3 1.2* 0.9* 1.1* 0.8* 41 801308 5.3 1.2* 1.2* 0.9* 1.1*42 801309 7.0 N.D. N.D. N.D. N.D. 43 801310 1.8 1.2* 1.2* 1.0* 1.0* 44801311 7.0 N.D. N.D. N.D. N.D. 41 801312 0.0 2.0 1.3 1.0 1.4 42 8013133.5 1.7* 1.1* 0.9* 0.9* 43 801314 0.0 1.9 1.2 1.0 2.9 44 801315 1.8 1.4*1.3* 1.0* 1.1* 36 801316 0.0 1.5 1.4 1.0 1.2 37 801298 3.5 1.4* 1.0*1.1* 1.3* 38 801299 5.3 1.2* 1.2* 1.0* 1.0* 39 801300 3.5 1.2* 1.1* 0.9*1.0* 40 801301 7.0 N.D. N.D. N.D. N.D. 38 801302 5.3 1.0* 1.1* 0.8* 0.8*39 801303 1.8 1.1* 1.0* 1.0* 1.1* 40 801304 4.8 14.3* 5.4* 2.2* 3.5* 38801305 5.3 3.6* 1.7* 1.6* 1.6* 39 801306 0.0 4.7 1.8 1.8 1.8 40 8066730.0 1.1 1.0 1.0 9.1 49 806674 0.0 1.0 1.0 0.8 8.2 50 806675 0.0 1.0 1.00.8 10.3 51 806676 0.0 1.0 1.0 0.8 1.5 49 806677 0.0 0.9 1.0 0.9 1.5 50806678 0.0 1.0 0.9 1.1 1.2 51 806679 0.0 1.2 1.1 1.1 1.3 49 806680 0.01.0 1.1 0.8 1.0 50 806681 0.0 1.0 1.0 0.9 1.4 51 806682 1.8 1.4* 1.2*1.0* 1.2* 49 806683 0.0 1.2 1.2 0.9 1.2 50 806684 0.0 1.2 1.1 0.9 2.0 51806685 0.0 1.0 0.9 0.8 1.5 52 806686 0.0 0.9 0.9 0.8 1.1 53 806687 0.01.0 1.0 0.8 1.0 54 806688 1.8 1.1* 1.0* 0.8* 1.8* 55 806689 0.0 1.1 1.00.8 1.3 52 806690 0.0 1.0 1.1 0.9 1.1 53 806691 5.3 N.D. N.D. N.D. N.D.54 802459 0.0 0.9 0.9 0.9 1.2 21 802460 5.3 1.1* 1.1* 1.0* 1.4* 45802461 3.5 1.4* 1.4* 0.9* 0.9* 46 802462 3.5 0.8* 0.9* 0.9* 1.1* 21802463 7.0 N.D. N.D. N.D. N.D. 45 802464 1.8 1.4* 1.2* 1.5* 1.4* 46802465 0.0 0.9 1.1 1.1 1.6 21 802466 1.8 1.0* 1.0* 1.0* 1.4* 45 8024671.3 4.9 2.4 2.7 2.8 46 802468 0.0 1.4 1.3 1.2 2.2 21 802469 0.0 1.0 1.01.0 1.2 45 802470 1.5 3.2 3.4 2.6 3.4 46 802471 0.0 0.9 0.9 0.9 1.2 47802472 3.5 1.6* 1.4* 0.9* 1.5* 48 802473 0.0 1.0 0.9 0.9 1.3 47 8024741.8 1.3* 1.2* 1.0* 1.8* 48 802475 7.0 N.D. N.D. N.D. N.D. 47 802476 1.81* 1.2* 0.8* 1.8* 48 802477 0.0 1.9 1.7 1.3 2.1 47 802478 3.5 1.4* 1.1*1.1* 1.3* 48

Example 13 Tolerability of Antisense Oligonucleotides Targeting HumanC9Orf72 in Rats

Sprague Dawley rats were separated into groups of 4 or 6 rats. Each ratin each group of rats was administered a single 3 mg intrathecal (IT)dose of the oligonucleotide indicated in the table below. At 3 hours andat 8 weeks following the IT dose, the movement of 7 different parts ofthe body was evaluated for each rat. The 7 body parts are (1) the rat'stail; (2) the rat's posterior posture; (3) the rat's hind limbs; (4) therat's hind paws; (5) the rat's forepaws; (6) the rat's anterior posture;(7) the rat's head. For each of the 7 different body parts, each rat wasgiven a sub-score of 0 if the body part was moving or 1 if the body partwas paralyzed. After each of the 7 body parts were evaluated, thesub-scores were summed for each rat and then averaged for each group.For example, if a rat's tail, head, and all other evaluated body partswere moving 3 hours after the 3 mg IT dose, it would get a summed scoreof 0. If another rat was not moving its tail 3 hours after the 3 mg ITdose but all other evaluated body parts were moving, it would receive ascore of 1. Saline treated rats generally receive a score of 0. A scoreof at the top end of the range would be suggestive of acute toxicity.Results are presented in the table below as the average score for eachtreatment group.

Animals were sacrificed at 8 weeks. The cortex and spinal cord werecollected from each animal, and RT-PCR was performed. Expression levelsof AIF1 were determined as a measure of inflammation. Expression levelsof (GFAP) were also determined as a measure of glial cell activation. Anasterisk indicates that the corresponding result is the average of 2-3mice. Results were normalized to Gapdh and are presented relative to PBScontrol (1.0) in the table below.

TABLE 16 Tolerability of antisense oligonucleotides targeting C9Orf72 inrats Score Score AIF1 GFAP 3 hours after 8 weeks after (spinal AIF1(spinal GFAP SEQ ID ISIS No. injection injection cord) (cortex) cord)(cortex) NO. 801287 2.5 0.0 1.9 1.2 1.3 1.3 33 801288 4.0 3.5 1.3* 1.3*1.2* 1.3* 34 806676 2.0 1.8 1.6* 1.5* 1.3* 2.5* 49 806679 1.2 0.0 1.41.3 1.3 1.8 49 806680 2.0 0.3 1.4 1.4 1.3 1.3 50 806690 1.3 0.0 1.5 1.41.3 2.0 53 802459 2.0 0.0 1.4 1.2 1.5 1.5 21 802473 2.0 2.3 1.8* 1.6*1.7* 1.6* 47

Example 14 Tolerability of Antisense Oligonucleotides Targeting HumanC9ORF72 in Non-Human Primates

Female cynomolgus monkeys (2-6 kg) were given 3 doses of 35 mg ofantisense oligonucleotide on days 1, 14, and 28 via intrathecal bolusinjection (1 mL slow bolus followed by 0.25 mL flush). Each treatmentgroup contained four monkeys. Two weeks after the final dose, animalswere sacrificed and RT-PCR was performed on various CNS tissues.Expression levels of AIF1 were determined as a measure of inflammationand expression levels of GFAP were determined as a measure of glial cellactivation. Results were normalized to GADPH and are presented relativeto PBS control (1.0) in the table below for ISIS No. 801287, 802459, and806679.

TABLE 17 Tolerability of antisense oligonucleotides targeting C9ORF72 incynomolgus monkeys AIF1 GFAP Brain Region 801287 802459 806679 801287802459 806679 Cervical spinal 1.0 0.9 0.9 1.1 1.0 1.1 cord Thoracicspinal 1.1 0.9 0.9 1.2 1.2 1.2 cord Temporal 1.0 1.0 1.5 1.2 1.1 1.7cortex Motor cortex 1.0 1.0 1.2 1.2 0.7 1.3 Lumbar spinal 1.2 0.9 0.91.3 0.9 1.1 cord Hippocampus 1.0 1.0 1.3 1.9 1.9 1.7 Frontal cortex 1.30.9 1.3 1.0 0.7 1.3

What is claimed is:
 1. A modified oligonucleotide according to thefollowing formula:

or a salt thereof.
 2. A modified oligonucleotide according to thefollowing formula:


3. The modified oligonucleotide of claim 1, which is a sodium salt ofthe formula.
 4. A pharmaceutical composition comprising the modifiedoligonucleotide of claim 1 and a pharmaceutically acceptable diluent orcarrier.
 5. The pharmaceutical composition of claim 4, wherein thepharmaceutically acceptable diluent is phosphate-buffered saline (PBS).6. The pharmaceutical composition of claim 4, wherein the pharmaceuticalcomposition consists essentially of the modified oligonucleotide andphosphate-buffered saline (PBS).
 7. A pharmaceutical compositioncomprising the modified oligonucleotide of claim 3 and apharmaceutically acceptable diluent or carrier.
 8. The pharmaceuticalcomposition of claim 7, wherein the pharmaceutically acceptable diluentis phosphate-buffered saline (PBS).
 9. The pharmaceutical composition ofclaim 7, wherein the pharmaceutical composition consists essentially ofthe modified oligonucleotide and phosphate-buffered saline (PBS).
 10. Apharmaceutical composition comprising the modified oligonucleotide ofclaim 2 and a pharmaceutically acceptable diluent or carrier.
 11. Thepharmaceutical composition of claim 10, wherein the pharmaceuticallyacceptable diluent is phosphate-buffered saline (PBS).
 12. Thepharmaceutical composition of claim 10, wherein the pharmaceuticalcomposition consists essentially of the modified oligonucleotide andphosphate-buffered saline (PBS).
 13. A compound comprising a modifiedoligonucleotide, wherein the modified oligonucleotide is a gapmerconsisting of a 5′ wing segment, a central gap segment, and a 3′ wingsegment, wherein: the 5′ wing segment consists of four 2′-O-methoxyethylnucleosides, the central gap segment consists of eightβ-D-deoxyribonucleosides, and the 3′ wing segment consists of six2′-O-methoxyethyl nucleosides; wherein the modified oligonucleotide hasthe nucleobase sequence 5′-GCCCCTAGCGCGCGACTC-3′ (SEQ ID NO: 33),wherein each cytosine is a 5-methylcytosine; and wherein theinternucleoside linkages of the modified oligonucleotide are, from 5′ to3′, soosssssssssoooss, wherein each s is a phosphorothioate linkage andeach o is a phosphodiester linkage.
 14. The compound of claim 13,comprising the modified oligonucleotide covalently linked to a conjugategroup.
 15. A pharmaceutical composition comprising the compound of claim13 and a pharmaceutically acceptable diluent or carrier.
 16. Thepharmaceutical composition of claim 15, wherein the pharmaceuticallyacceptable diluent is phosphate-buffered saline (PBS).
 17. Thepharmaceutical composition of claim 15, wherein the pharmaceuticalcomposition consists essentially of the compound and phosphate-bufferedsaline (PBS).
 18. A pharmaceutical composition comprising the compoundof claim 14 and a pharmaceutically acceptable diluent or carrier. 19.The pharmaceutical composition of claim 18, wherein the pharmaceuticallyacceptable diluent is phosphate-buffered saline (PBS).
 20. Thepharmaceutical composition of claim 18, wherein the pharmaceuticalcomposition consists essentially of the compound and phosphate-bufferedsaline (PBS).